Rab7b participation on the TLR4 (Toll-like receptor) endocytic pathway in Shiga toxin-associated Hemolytic Uremic Syndrome (HUS).

BACKGROUND: The inflammatory response of the host to Shiga toxin and/or lipopolysaccharide (LPS) of Escherichia coli (E. coli) is included in (HUS). The TLR4-LPS complex is internalized and TLR4 induced inflammatory signaling is stopped by targeting the complex for degradation. Rab7b, a small guanosine triphosphatase (GTPase) expressed in monocytes, regulates the later stages of the endocytic pathway. OBJECTIVE: we studied the Rab7b participation on the TLR4 endocytic pathway and its effect on monocyte cytokine production along the acute course of pediatric Shiga toxin-associated HUS. METHODS AND RESULTS: Monocytes were identified according to their positivity in CD14 expression. Surface TLR4 expression in monocytes from 18 HUS patients significantly increased by day 1 to 6, showing the highest increase on day 4 compared to monocytes of 10 healthy children. Significant higher surface TLR4 expression was accompanied by increased proinflammatory intracellular cytokines, tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6). In contrast, after these time points, surface TLR4 expression and intracellular TNF-α levels, returned to near control levels after 10 days. Furthermore, confocal immunofluorescence microscopy proved colocalization of increased intracellular TLR4/Rab7b determined by Pearson's coefficient in monocytes from HUS patients from day 1 on the highest colocalization of both proteins by day 4. Decreased TLR4/Rab7b colocalization was shown 10 days after HUS onset. CONCLUSION: The colocalization of TLR4 and Rab7b allows us to suggest Rab7b participation in the control of the TLR4 endocytic pathway in HUS patient monocytes. A consequential fall in cytokine production throughout the early follow up of HUS is demonstrated.

Urinary Renin in Patients and Mice With Diabetic Kidney Disease.

In patients with diabetic kidney disease (DKD), plasma renin activity is usually decreased, but there is limited information on urinary renin and its origin. Urinary renin was evaluated in samples from patients with longstanding type I diabetes mellitus and mice with streptozotocin-induced diabetes mellitus. Renin-reporter mouse model (Ren1d-Cre;mT/mG) was made diabetic with streptozotocin to examine whether the distribution of cells of the renin lineage was altered in a chronic diabetic environment. Active renin was increased in urine samples from patients with DKD (n=36), compared with those without DKD (n=38; 3.2 versus 1.3 pg/mg creatinine; P<0.001). In mice with streptozotocin-induced diabetes mellitus, urine renin was also increased compared with nondiabetic controls. By immunohistochemistry, in mice with streptozotocin-induced diabetes mellitus, juxtaglomerular apparatus and proximal tubular renin staining were reduced, whereas collecting tubule staining, by contrast, was increased. To examine the role of filtration and tubular reabsorption on urinary renin, mice were either infused with either mouse or human recombinant renin and lysine (a blocker of proximal tubular protein reabsorption). Infusion of either form of renin together with lysine markedly increased urinary renin such that it was no longer different between nondiabetic and diabetic mice. Megalin mRNA was reduced in the kidney cortex of streptozotocin-treated mice (0.70±0.09 versus 1.01±0.04 in controls, P=0.01) consistent with impaired tubular reabsorption. In Ren1d-Cre;mT/mG with streptozotocin-induced diabetes mellitus, the distribution of renin lineage cells within the kidney was similar to nondiabetic renin-reporter mice. No evidence for migration of cells of renin linage to the collecting duct in diabetic mice could be found. Renin mRNA in microdissected collecting ducts from streptozotocin-treated mice, moreover, was not significantly different than in controls, whereas in kidney cortex, largely reflecting juxtaglomerular apparatus renin, it was significantly reduced. In conclusion, in urine from patients with type 1 diabetes mellitus and DKD and from mice with streptozotocin-induced diabetes mellitus, renin is elevated. This cannot be attributed to production from cells of the renin lineage migrating to the collecting duct in a chronic hyperglycemic environment. Rather, the elevated levels of urinary renin found in DKD are best attributed to altered glomerular filteration and impaired proximal tubular reabsorption.

Physiological Functions and Regulation of the Na+/H+ Exchanger [NHE1] in Renal Tubule Epithelial Cells.

The sodium-hydrogen exchanger isoform-1 [NHE1] is a ubiquitously expressed plasma membrane protein that plays a central role in intracellular pH and cell volume homeostasis by catalyzing an electroneutral exchange of extracellular sodium and intracellular hydrogen. Outside of this important physiological function, the NHE1 cytosolic tail domain acts as a molecular scaffold regulating cell survival and actin cytoskeleton organization through NHE1-dependent signaling proteins. NHE1 plays main roles in response to physiological stress conditions which in addition to cell shrinkage and acidification, include hypoxia and mechanical stimuli, such as cell stretch. NHE1-mediated modulation of programmed cell death results from the exchanger-mediated changes in pHi, cell volume, and/or [Na+]I; and, it has recently become known that regulation of cellular signaling pathways are involved as well. This review focuses on NHE1 functions and regulations. We describe evidence showing how these structural actions integrate with ion translocation in regulating renal tubule epithelial cell survival.

Granulomatosis with polyangiitis: rapidly progressive necrotizing glomerulonephritis in a pediatric patient.

Granulomatosis with polyangiitis (GPA) is associated with a broad range of clinical manifestations including renal disease. It is a systemic vasculitis that is rarely encountered in children. We present a 14-year-old girl who suffered from pharyngitis 1 week before admittance to hospital. She was admitted for macroscopic hematuria and oliguria, under the possibility of nephritic syndrome. Renal failure with rapidly progressive glomerulonephritis occurred within 24 hours. Immunologic tests showed the presence of type-C anti-neutrophil cytoplasmic antibodies (c-ANCA with antiproteinase 3 specificity) and renal biopsy revealed pauci-immune crescentic focal necrotizing glomerulonephritis. Treatment including methylprednisolone and cyclophosphamide intravenous pulses allowed renal recovery after 3 weeks. The clinical, hematological, and biochemical parameters improved substantially, achieving remission. Granulomatosis with polyangiitis, although rare in children, should be considered in the above clinical scenario. This case underlines that knowledge of renal histology diagnosis and early aggressive immunosuppressive therapy are essential for the management of these patients.

Toll-like receptor 4 expression on circulating leucocytes in hemolytic uremic syndrome.

Lipopolysaccharide stimulation of toll-like receptor 4 (TLR4) activates signal transduction pathways leading to proinflammatory cytokine secretion. We investigated TLR4 surface receptor expression on peripheral blood neutrophils and monocytes and their ability to modulate inflammatory cytokine release in 15 patients 1, 3, and 10 days after hemolytic uremic syndrome (HUS) onset. Seven patients with Escherichia coli (EHEC)-associated diarrhea and seven healthy controls were also studied. Isolated leucocytes from HUS-onset patients exhibited significantly higher messenger RNA (mRNA) TLR4 expression than controls. Moreover, TLR4 protein expression on neutrophils, determined by flow cytometry, was upregulated, driving dependent proinflammatory cytokine, tumor necrosis factor alpha (TNF-α), and interleukin 8 (IL-8) increase, and decreased anti-inflammatory IL-10 release at HUS onset compared with patients with EHEC diarrhea and controls. TLR4 expression on neutrophils was positively correlated with serum TNF-α levels. Conversely, significant reduction of neutrophil TLR4 receptor expression and lack of cytokine-responsive element activation was shown in patients 3 and 10 days after HUS onset. No differences were demonstrated in TLR4 receptor expression on monocytes among the studied groups. Our results suggest TLR4 expression may be differently regulated on neutrophils and monocytes. They could be dynamically modulated across the early development of HUS on neutrophils, resulting in negative regulation preceded by TLR4 overactivation.

Adiponectin in children on peritoneal dialysis: relationship to insulin resistance and nutritional status.

AIM: To study whether adiponectin and resistin serum concentrations in children on peritoneal dialysis (PD) were related to insulin resistance (IR) and anthropometric parameters of nutritional status, 11 PD patients, 9 chronic kidney disease (CKD) patients and 10 healthy children were studied. METHODS: Glucose and insulin were measured during the oral glucose tolerance test. Levels of adiponectin and resistin were evaluated by ELISA, insulin by RIA. RESULTS: In CKD patients, higher homeostasis model assessment-insulin resistance (HOMA-IR), fasting and 2-hour serum insulin levels were shown compared to control and to PD patients. Body mass index (BMI) and body fat content were severely decreased while serum adiponectin levels were significantly higher in PD patients relative to controls. No differences among groups were shown in resistin levels. On regression modeling, inverse independent associations were observed between adiponectin with percentile BMI, weight and height z-score, and with body fat content. In contrast, no relationship was found between adiponectin and IR parameters. In multiple regression analysis, adiponectin was negatively correlated with BMI. A negative association of adiponectin and resistin with glomerular filtration rate was also shown. CONCLUSION: A role for adiponectin in terms of its association with clinical wasting parameters in PD pediatric patients might be suggested.

Altered renal expression of Na(+) transporters and ROMK in protein-deprived rats.

Potassium depletion has been associated with altered sodium reabsorption in tubule segments. We studied if the altered abundance of Na(+) transporters and ROMK are associated with distal potassium secretion that contributes to the development of hypokalemia in protein-deprived rats. After weaning, Wistar rats were fed with a low-protein diet (8%, LP) for 14 days and then recovered with a normal-protein (NP) diet (24%, RP). An age-matched control group was fed with an NP diet (24%, NP). We showed hypokalemia, lower glomerular filtration rate and higher FEK(+) in the LP group. Immunoblotting revealed that the type 3 Na(+)/H(+) exchanger in the cortex was decreased in the LP group. However, the type 2 Na(+)-K(+)-2Cl(-) cotransporter was increased in the outer stripe of the outer medulla in the LP group. The abundance of the aldosterone-regulated Na(+)-Cl(-) cotransporter (NCC) and epithelial Na(+) channel (ENaC) was higher in the LP group and was associated with higher plasma aldosterone level. ROMK protein levels were increased. Na(+)/K(+)-ATPase protein levels were the same in both groups. After the recovery period, the expression of Na(+) transporters and ROMK returned to control values. We conclude that increased expression of NCC, ENaC subunits, and ROMK contributed to distal potassium secretion leading to enhanced potassium excretion, which may explain the hypokalemia resulting from LP feeding. A role of aldosterone may be suggested.

Na+/K+ -ATPase stabilization by Hsp70 in the outer stripe of the outer medulla in rats during recovery from a low-protein diet.

A low-protein (LP) diet induces injury from energy depletion in renal epithelial cells. Overexpression of heat-shock proteins has been implicated in the restoration of the cytoskeletal anchorage of Na(+)/K(+)-ATPase. We tested if Hsp70 stabilizes renal Na(+)/K(+)-ATPase attachment to the cytoskeleton from the cortex and the outer stripe of the outer medulla (OSOM) in rats during recovery from a LP diet. Rats were fed with a LP diet (8% protein) for 14 days, and then the rats were recovered with a 24% protein (RP) diet. The control group received a 24% protein (NP) diet. Increased Na(+)/K(+)-ATPase dissociation was demonstrated in soluble fraction from OSOM with lower ATP content as a result of LP diet vs NP. Meanwhile, decreased Hsp70 levels in the same fraction were shown. Translocation of Hsp70 to the cytoskeletal injured fraction associated with stabilization of Na(+)/K(+)-ATPase was shown in OSOM from LP after in vitro co-incubation of the cytoskeletal fraction of LP and non-cytoskeletal fraction of RP. These effects were abolished by the addition of the anti-Hsp70 antibody. Absence of Na(+)/K(+)-ATPase detachment from its cytoskeletal anchorage was demonstrated in proximal duct segments from cortex in LP. Co-immunoprecipitation showed that the amount of Na(+)/K(+)-ATPase co-precipitating with Hsp70 increased in the OSOM as a result of the LP diet. In the cortex tissues from rats fed the LP and the RP diet, the interaction of both proteins were similar to the control groups. Our results indicate that Hsp70 has a critical role in protecting the integrity of the cytoskeletal anchorage of Na(+)/K(+)-ATPase during recovery from ATP-depleted injury resulting from LP in OSOM.

Cytoprotective role of nitric oxide associated with Hsp70 expression in neonatal obstructive nephropathy.

Nitric oxide (NO) has emerged as an important endogenous inhibitor of apoptosis. In this study, we postulated that the mechanism of apoptosis inhibition by NO would include stimulation of heat shock protein 70 (Hsp70) expression. Rats were subjected to unilateral ureteral obstruction (UUO) or sham operation, and kidneys were harvested 5 and 14 days after obstruction. After 14 days of obstruction, decreased endogenous NO and lower inducible nitric oxide synthase (iNOS) expression at mRNA and protein levels associated with downregulation of Hsp70 protein expression were shown in apoptosis induction, regulated by mitochondrial signal pathway, through the increased pro-apoptotic ratio Bax/BcL(2) and consequently caspase 3 activity. Conversely, 5 days after kidney obstruction, increased Hsp70 expression linked to increase NO and iNOS expression at transcriptional and post-transcriptional levels with absence of apoptotic response, were demonstrated. In obstructed neonatal rats, in vivo administration of l-Arginine induced heat shock protein 70 (Hsp70) expression, which was associated with cytoprotection from apoptosis and transiently decreased nicotinamide adenine dinucleotide phosphate reduced form (NADPH) oxidase activity. Opposite effects were obtained after nitro L-Arginine methyl ester (L-NAME) treatment. The interaction between B-cell lymphoma 2 anti-apoptotic members (BcL(2)) and Hsp70 in the presence of L-Arginine and L-NAME, was determined by coimmunoprecipitation. Binding of BcL(2) and Hsp70 increased after L-Arginine administration. These findings suggest that NO can produce resistance to obstruction-induced cell death by mitochondrial apoptotic pathway, through the induction of Hsp70 expression, in neonatal unilateral ureteral obstruction.

Renal caveolin-1 expression in children with unilateral ureteropelvic junction obstruction.

Caveolae are plasma membrane invaginations that contain a variety of signal transduction molecules and receptors for growth factors and cytokines. This study was performed to examine the in vivo expression and localization of caveolin-1 in kidneys from 19 children who underwent surgery release of ureteropelvic junction obstruction (UPJO) in relation to renal function and degree of tubulointerstitial fibrosis. Renal biopsies were carried out at the time of surgery for obstruction release. Kidney tissue from children of similar age removed because of carcinoma was used as control. Expression of caveolin-1 at the protein level in renal tissue and urine was demonstrated in patients with technetium 99 m labeled diethylene triamine pentaacetate ((99)Tc DTPA) renal scan 28.8+/-2% and increased tubular interstitial fibrosis in seven patients at the time of obstruction release. Colocalization staining of AT(1) angiotensin II receptor with caveolin-1 in basolateral membrane of epithelial tubule cells, enhanced AT(1) messenger ribonucleic acid (mRNA) and decreased endothelial nitric oxide synthase (eNOS), were shown in these patients. In contrast, absence of association of caveolin-1 with AT(1) receptor expression in proximal and collecting tubule membranes with AT(1) receptor mRNA and eNOS mRNA expression near control were demonstrated in 12 patients, with (99)Tc DTPA renal scan 39.7+2.1% and no evidence of tubulointerstitial fibrosis. From our results, the role of caveolin-1 as a factor contributing to the severity of the tubulointerstitial process resulting from obstructive nephropathy could be suggested.

Heat shock protein 70 expression is associated with inhibition of renal tubule epithelial cell apoptosis during recovery from low-protein feeding.

The cellular stress response can mediate cellular protection through expression of heat shock protein (Hsp70), which can interfere with the process of apoptotic cell death. Factors regulating renal epithelial cell apoptosis include angiotensin II. In the present study, we have examined the relationship between the Hsp70 expression and the apoptotic pathway in the kidneys from low-protein-fed rats (8% protein). The possible cytoprotective role of Hsp70 has been evaluated during low-protein feeding and after reincorporation of 24% protein in the diet. The effect of angiotensin II AT1 receptor inhibition has also been studied. Rats were fed with a low-protein (LP) diet (8% protein) for 14 days, and then the animals were recovered by means of a normal protein diet (24% protein) (RP) for 14, 21, and 30 days, and control rats received 24% protein (NP) in the diet. LP and NP rats treated with Losartan (10 mg/kg) were also evaluated. The following methods were performed on the kidneys: terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling assay for apoptosis, reverse transcriptase-polymerase chain reaction assay for AT1, Bax, and Bcl-2 messenger ribonucleic acid (mRNA) expression, and immunohistochemical and Western blot for Hsp70 and caspase 3 protein expression and activity. In the LP group, the cells of the medullary ducts (MDs) showed increased apoptosis associated with weak immunoreaction for Hsp70 and decreased Hsp70 protein levels. In these animals, enhanced proapoptotic ratio Bax/Bcl-2 linked to decreased procaspase 3 protein levels with increased caspase 3 activation were demonstrated. A cytoprotection attributed to Hsp70 could be noted in the RP rats after 21 days of reincorporation of the normal diet, and in the LP-fed group treated with Losartan. In these cases, the MD cells displayed decreased apoptosis and increased Hsp70 expression in colocalization staining, and high Hsp70 levels in cytosolic fraction. A decreased proapoptotic ratio Bax/Bcl-2, associated with increased Bcl-2 mRNA, was also observed. Our results provide evidence for an antiapoptotic, cytoprotective effect of Hsp70 in kidney MD cells of rats with LP intake, when the animals were recovered with 24% protein in diet and after angiotensin II AT1 receptor inhibition. Angiotensin II seems to play a role in the pathogenesis of tubule epithelial cell apoptosis during LP feeding.

H+_Atpasa Vacuolar Renal. Fisiología y Regulación / Vacuolar H (+)_ATPase renal. Physiologi and Regulation

Las H+ _ATPasas vacuolares son complejos proteicos ubicuos con múltiples subunidades que incluyen un dominio catalítico V1 compuesto por proteínas periféricas que hidrolizan adenosina trisfosfato (ATP) y proveen energía a la bomba de H+ a través de un segundo dominio transmembrana Vº en contra de importantes gradientes. Estas H+_ATPasas vacuolares, que intervienen en la translocación de protones, son responsables en células eucariotas de la acidificación de las organelas intracelulares y de la acidificación de los espacios luminales e intersticiales adyacentes a las membranas plasmáticas celulares. Mutaciones en los genes que codifican las subunidades de H+ATPasa vacuolar específicas de células intercalares de ríñón; Vºa4 y V1B1, causan el síndrome de acidosis tubular distal renal. Esta revisión se focalizará en función, regulación y en el rol de H+ATPasa vacuolar en fisiología renal. La localización de H+ _ATPasa en el rión y su rol en la regulación de pHintracelular, transporte de protones y la homostsis del ácido base, será discutida.

H-ATPase activity in collecting duct segments in protein-deprived rats - role of angiotensin II on its regulation.

BACKGROUND: Enhanced expression of the genes that encode for components of the renin-angiotensin system (RAS) has been exhibited in low-protein-fed (LP) rats. We examined distal proton secretion in LP rats through the activity of H(+)-ATPase on microdissected CCD, OMCD and IMCD segments. The effect of angiotensin II AT(1) receptor inhibition and protein recovery (24% protein) on H(+)-ATPase activity was studied. METHODS: Bafilomycin-sensitive H(+)-ATPase activity on CCD, OMCD and IMCD segments of LP (protein 8%) and control rats CP (protein 24%) was evaluated. We examined the levels of mRNA expression of RAS components: angiotensin-converting enzyme (ACE), angiotensinogen and angiotensin II AT(1) expression; AT(1 )receptor binding and distribution were determined by quantitative autoradiography. RESULTS: Increased ACE and AT(1) mRNA expression was found in cortex and medulla of LP compared to NP rats. AT(1) receptor binding density was significantly reduced in renal cortex and inner stripe of the outer medulla of LP compared to NP rats. Minimal radioligand binding was shown in inner medulla of LP. Whole kidney expression of angiotensinogen was unaltered in LP. H(+)-ATPase activity significantly decreased in IMCDs and OMCDs of LP. The inhibitory effect of LP was abolished when OMCD segments were incubated for 60 min in the presence of losartan 10(-6) to 10(-8)M. There was no effect of losartan concentrations from 10(-6) to 10(-8) M on IMCDs. Similar results were observed on H(+)-ATPase activity in OMCD and IMCD segments after readministration of 24% protein in the diet. CONCLUSION: Both the recovery of H(+)-ATPase activity in OMCD segments induced by losartan and the increased expression of AT(1 )receptor suggest angiotensin II modulation of proton ATPase activity on this duct segments in LP rats. Intense compromise of proton secretion through the continued H(+)-ATPase inhibition in IMCDs from LP was shown.

Role of endogenous nitric oxide in unilateral ureteropelvic junction obstruction in children.

BACKGROUND: Obstructive nephropathy leads to tubulointerstitial fibrosis and loss of renal function. Nitric oxide has been shown to have antifibrotic properties. We examined nitric oxide synthase (NOS) activity and expression in kidneys from children who underwent surgery release of unilateral ureteropelvic junction (UPJ) obstruction in relation to clinical and histologic parameters. METHODS: NOS activity and the expression of NOS isoforms measured at the mRNA level by reverse transcription-polymerase chain reaction (RT-PCR) assay were determined in tissue obtained by biopsy from obstructed kidneys of 18 children at the time of pyeloplasty. Tissue from kidneys removed because of various malignancies were issued as control. RESULTS: A significant increase in calcium/calmodulin-independent NOS activity (iNOS) and iNOS mRNA expression was found in the medulla of obstructed kidneys. Calcium/calmodulin-dependent NOS activity (cNOS) and endothelial (eNOS) mRNA, by contrast, were increased in the cortex from obstructed kidneys. A role of tumor necrosis factor-alpha (TNF-alpha) on enhanced iNOS was suggested by the finding of increased urine levels in obstructed pelvis. Increased interstitium macrophage number, by immunolabeling of CD68, was related to the delay in obstruction release and to decreased glomerular filtration rate (GFR) at surgery. A positive linear relationship was found between cNOS activity in cortex and creatinine clearance. The degree of interstitial fibrosis correlated negatively with cNOS activity in cortex. CONCLUSION: In kidneys from children with UPJ obstruction an increased activity and expression of iNOS in medulla and cNOS-dependent eNOS in cortex were demonstrated. A role of cNOS in modulating GFR and interstitial fibrosis can be suggested. Prolonged UPJ obstruction would lead to a worsened prognosis on renal injury.