The Relationship between Urinary Stones and Gut Microbiomeby 16S Sequencing.

OBJECTIVE: To understand the relationship between urinary stones and the gut microbiome and to screen for microbial species that may be involved in stone formation. METHODS: Stool samples were collected from patients with urolithiasis and healthy patients between March and December 2017. The samples were analyzed by 16S sequencing to determine differences in the microbiome profiles between the two groups. The mouse model was established and was divided into two groups. Fecal samples were collected from the mice before gavage and three weeks postgavage for microbiome analysis. The microbial population of each group was analyzed to screen for microbial species that may affect the formation of urinary stones. Differences in the number of crystals in the renal tubules of the mice were examined by necropsy. RESULTS: The microbial composition was different between urolithiasis patients and healthy controls. The urolithiasis patients had significantly reduced microbial abundance; however, increased proportions of Bacteroidetes and Actinobacteria were detected compared to healthy controls. Furthermore, the abundance of Alistipesindistinctus and Odoribactersplanchnicus was significantly increased in the urolithiasis patients compared to the healthy controls. In addition, the incidence of urolithiasis was much higher in the experimental mouse group (stone solution + urolithiasis patient stool) than in the control mouse group. However, the microbial abundance before gavage was not significantly different from that seen three weeks postgavage. CONCLUSION: Theurolithiasis patients in this study had a different gut microbiome when compared with that of healthy individuals. The altered microbiome increased the rate of crystal formation in renal tubules and accelerated urinary stone formation in the mouse model of urolithiasis.

Regulation of Fn14 stability by SCFFbxw7α during septic acute kidney injury.

Acute kidney injury (AKI) initiated by sepsis remains a thorny problem despite recent advancements in its clinical management. Having been found to be activated during AKI, fibroblast growth factor-inducible molecule 14 (Fn14) may be a potential therapeutic target because of its involvement in the molecular basis of injury. Here, we report that LPS induces apoptosis of mouse cortical tubule cells mediated by Fn14, for which simultaneous Toll-like receptor (TLR)4 activation is required. Mechanistically, TLR4 activation by lipopolysaccharide, through disassociating E3 ligase SCFFbxw7α from Fn14, dismantles Lys48-linked polyubiquitination of Fn14 and stabilizes it. Pharmacological deactivation of Fn14 with monoclonal antibody ITEM-2 provides effective protection against lethal sepsis and AKI in mice. Our study underscores an adaptive mechanism whereby TLR4 regulates SCFFbxw7α-dependent Fn14 stabilization during inflammatory tubular damage and further supports investigation of targeting Fn14 in clinical trials of patients with septic AKI.

Persistent Leptospiruria in Five Dogs Despite Antimicrobial Treatment (2000-2017).

In dogs with leptospirosis, doxycycline therapy is recommended as the preferred therapy for its ability to eliminate the organism from all tissues, including the renal tubules. Elimination of organisms from the renal tubules terminates leptospiruria and prevents transmission of the organism. This report describes the discovery of persistent leptospiruria in the face of therapy with doxycycline in four dogs and enrofloxacin in one dog. Leptospiruria was confirmed by polymerase chain reaction testing for pathogenic leptospires in all five dogs. In two dogs, leptospiruria resolved after a change in therapy to enrofloxacin. In three dogs, doxycycline and/or enrofloxacin were ineffective at eliminating leptospiruria, which then resolved after therapy with clarithromycin. Pet owners could be at risk as persistent leptospiruria poses a potential zoonotic risk. The potential reasons for persistent leptospiruria as demonstrated by polymerase chain reaction testing are discussed.

Androgen exposure potentiates formation of intratubular communities and renal abscesses by Escherichia coli.

Females across their lifespan and certain male populations are susceptible to urinary tract infections (UTI). The influence of female vs. male sex on UTI is incompletely understood, in part because preclinical modeling has been performed almost exclusively in female mice. Here, we employed established and new mouse models of UTI with uropathogenic Escherichia coli (UPEC) to investigate androgen influence on UTI pathogenesis. Susceptibility to UPEC UTI in both male and female hosts was potentiated with 5α-dihydrotestosterone, while males with androgen receptor deficiency and androgenized females treated with the androgen receptor antagonist enzalutamide were protected from severe pyelonephritis. In androgenized females and in males, UPEC formed dense intratubular, biofilm-like communities, some of which were sheltered from infiltrating leukocytes by the tubular epithelium and by peritubular fibrosis. Abscesses were nucleated by small intratubular collections of UPEC first visualized at five days postinfection and briskly expanded over the subsequent 24 hours. Male mice deficient in Toll-like receptor 4, which fail to contain UPEC within abscesses, were susceptible to lethal dissemination. Thus, androgen receptor activation imparts susceptibility to severe upper-tract UTI in both female and male murine hosts. Visualization of intratubular UPEC communities illuminates early renal abscess pathogenesis and the role of abscess formation in preventing dissemination of infection. Additionally, our study suggests that androgen modulation may represent a novel therapeutic route to combat recalcitrant or recurrent UTI in a range of patient populations.

Association of brucellosis with renal tubular and glomerular damage in children in Turkey.

Brucellosis is a multisystem disease that may present with a broad spectrum of clinical manifestations. Until now, no studies have been performed on renal tubular disorders in patients with brucellosis. The present study aims to investigate renal tubular disorders in patients with brucellosis. This prospective case-control study includes a total of 31 brucellosis patients (Group 1) and 30 healthy controls (Group 2) matched for age and sex. Renal tubular functions of children who were diagnosed as having brucellosis in outpatient pediatric clinics were evaluated. First-morning urine samples were collected from Group 1 and Group 2 at the same time. Urea, creatinine, potassium, sodium, and phosphorus were determined in serum and urine by an autoanalyzer. Tubular reabsorption and excretion of urine electrolytes were calculated using the related formulas. Patients with brucellosis had significantly lower levels of tubular reabsorption of phosphorus and serum phosphorus than those of the control group. Furthermore, urine sodium and serum potassium levels and fractionated sodium excretion of brucellosis patients were significantly higher than healthy control group. Estimated glomerular filtration rate was remarkably higher in the patient group (P < 0.001).We concluded that tubular and glomerular functional parameters demonstrate deterioration in patients with brucellosis compared to those in healthy participants.

Proteomic analysis of urine exosomes reveals renal tubule response to leptospiral colonization in experimentally infected rats.

BACKGROUND: Infectious Leptospira colonize the kidneys of reservoir (e.g. rats) and accidental hosts such as humans. The renal response to persistent leptospiral colonization, as measured by urinary protein biosignatures, has not been systematically studied. Urinary exosomes--bioactive membrane-bound nanovesicles--contain cell-state specific cargo that additively reflect formation all along the nephron. We hypothesized that Leptospira-infection will alter the content of urine exosomes, and further, that these Leptospira-induced alterations will hold clues to unravel novel pathways related to bacterial-host interactions. METHODOLOGY/PRINCIPAL FINDINGS: Exosome protein content from 24 hour urine samples of Leptospira-infected rats was compared with that of uninfected rats using SDS-PAGE and liquid chromatography/tandem mass spectrometry (LC-MS/MS). Statistical models were used to identify significantly dysregulated proteins in Leptospira-infected and uninfected rat urine exosomes. In all, 842 proteins were identified by LC-MS/MS proteomics of total rat urine and 204 proteins associated specifically with exosomes. Multivariate analysis showed that 25 proteins significantly discriminated between uninfected control and infected rats. Alanyl (membrane) aminopeptidase, also known as CD13 topped this list with the highest score, a finding we validated by Western immunoblotting. Whole urine analysis showed Tamm-Horsfall protein level reduction in the infected rat urine. Total urine and exosome proteins were significantly different in male vs. female infected rats. CONCLUSIONS: We identified exosome-associated renal tubule-specific responses to Leptospira infection in a rat chronic colonization model. Quantitative differences in infected male and female rat urine exosome proteins vs. uninfected controls suggest that urine exosome analysis identifies important differences in kidney function that may be of clinical and pathological significance.

Direct acute tubular damage contributes to Shigatoxin-mediated kidney failure.

The pathogenesis and therapy of Shigatoxin 2 (Stx2)-mediated kidney failure remain controversial. Our aim was to test whether, during an infection with Stx2-producing E. coli (STEC), Stx2 exerts direct effects on renal tubular epithelium and thereby possibly contributes to acute renal failure. Mice represent a suitable model because they, like humans, express the Stx2-receptor Gb3 in the tubular epithelium but, in contrast to humans, not in glomerular endothelia, and are thus free of glomerular thrombotic microangiopathy (TMA). In wild-type mice, Stx2 caused acute tubular dysfunction with consequent electrolyte disturbance, which was most likely the cause of death. Tubule-specific depletion of Gb3 protected the mice from acute renal failure. In vitro, Stx2 induced secretion of proinflammatory cytokines and apoptosis in human tubular epithelial cells, thus implicating a direct effect of Stx2 on the tubular epithelium. To correlate these results to human disease, kidney biopsies and outcome were analysed in patients with Stx2-associated kidney failure (n = 11, aged 22-44 years). The majority of kidney biopsies showed different stages of an ongoing TMA; however, no glomerular complement activation could be demonstrated. All biopsies, including those without TMA, showed severe acute tubular damage. Due to these findings, patients were treated with supportive therapy without complement-inhibiting antibodies (eculizumab) or immunoadsorption. Despite the severity of the initial disease [creatinine 6.34 (1.31-17.60) mg/dl, lactate dehydrogenase 1944 (753-2792) U/l, platelets 33 (19-124)/nl and haemoglobin 6.2 (5.2-7.8) g/dl; median (range)], all patients were discharged after 33 (range 19-43) days with no neurological symptoms and no dialysis requirement [creatinine 1.39 (range 0.84-2.86) mg/dl]. The creatinine decreased further to 0.90 (range 0.66-1.27) mg/dl after 24 months. Based on these data, one may surmise that acute tubular damage represents a separate pathophysiological mechanism, importantly contributing to Stx2-mediated acute kidney failure. Specifically in young adults, an excellent outcome can be achieved by supportive therapy only.

Effects of Escherichia coli subtilase cytotoxin and Shiga toxin 2 on primary cultures of human renal tubular epithelial cells.

Shiga toxin (Stx)-producing Escherichia coli (STEC) cause post-diarrhea Hemolytic Uremic Syndrome (HUS), which is the most common cause of acute renal failure in children in many parts of the world. Several non-O157 STEC strains also produce Subtilase cytotoxin (SubAB) that may contribute to HUS pathogenesis. The aim of the present work was to examine the cytotoxic effects of SubAB on primary cultures of human cortical renal tubular epithelial cells (HRTEC) and compare its effects with those produced by Shiga toxin type 2 (Stx2), in order to evaluate their contribution to renal injury in HUS. For this purpose, cell viability, proliferation rate, and apoptosis were assayed on HRTEC incubated with SubAB and/or Stx2 toxins. SubAB significantly reduced cell viability and cell proliferation rate, as well as stimulating cell apoptosis in HRTEC cultures in a time dependent manner. However, HRTEC cultures were significantly more sensitive to the cytotoxic effects of Stx2 than those produced by SubAB. No synergism was observed when HRTEC were co-incubated with both SubAB and Stx2. When HRTEC were incubated with the inactive SubAA272B toxin, results were similar to those in untreated control cells. Similar stimulation of apoptosis was observed in Vero cells incubated with SubAB or/and Stx2, compared to HRTEC. In conclusion, primary cultures of HRTEC are significantly sensitive to the cytotoxic effects of SubAB, although, in a lesser extent compared to Stx2.

Histopathological kidney alterations in rats naturally infected with Leptospira / Alteraciones histopatológicas en ratas naturalmente infectadas con Leptospira

Introduction: Histopathological changes by Leptospira in naturally infected rodent reservoirs have been poorly described. Objective: The aim of the current study is to describe renal histopathology associated with leptospirosis infection of naturally infected rodents captured in the urban area of the city of Medellin, Colombia. Materials and methods: We performed hematoxilin-eosin (H-E) on kidney samples collected from 254 captured rodents. The positive samples were processed by Warthin Starry (W-S) staining and PCR- LipL 32. Results: Fifty one rodent kidneys showed H-E histopathological changes that consisted of inflammatory infiltrate with lympho-plasmocitary cells and histiocytes. We performed W-S staining and PCR- LipL 32 to 67 kidney samples, including the 51 that had shown detectable changes by H-E and 16 (8%) of 203 rodents with negative results. Eight of the samples that tested positive for H-E (15.7%) were also positive for W-S staining. All negative for H-E were also negative for W-S staining. Of the W-S positive samples also tested for culture only three tested positive for both. Additionally, 47 (92.1%) samples positive for H-E were positive for PCR; while eleven of the 16 (68.8%) negative for H-E were positive for PCR. The samples positive for PCR were subsequently tested for culture and 11 (23.4%) were positive. Seven samples were positive for PCR and W-S and three were positive for PCR, W-S and culture. All of the PCR- LipL 32 fragments were sequenced and showed specific amplicons for L. interrogans . Conclusions: The Leptospira infection was confirmed in all of the animals tested. The only histological kidney lesion attributable to leptospiral infection in the reservoir was interstitial nephritis.

Introducción. Los hallazgos histopatológicos ocasionados por Leptospira spp. han sido poco estudiados en poblaciones de roedores naturalmente infectados. Objetivo. Describir la histopatología renal asociada con las infecciones naturalmente adquiridas en un grupo de roedores capturados en el área urbana de Medellín, Colombia. Materiales y métodos. Se llevaron a cabo coloraciones de hematoxilina y eosina de los riñones de 254 roedores recolectados en el área de estudio. Las muestras positivas se procesaron con la coloración de Warthin-Starry y mediante reacción en cadena de la polimerasa (PCR)-LipL32. Results. Se observaron cambios histopatológicos con hematoxilina y eosina en 51 riñones de roedores, que consistieron en infiltrado inflamatorio con linfoplasmocitos e histiocitos. Se utilizó coloración de Warthin-Starry y PCR-LipL32 en 67 muestras de riñón que incluyeron las 51 muestras que tuvieron cambios detectables por hematoxilina y eosina y 16 de 203 (8 %) muestras con resultados negativos. Ocho de las muestras positivas por hematoxilina y eosina (15,7 %) también fueron positivas por la coloración de Warthin-Starry. Las muestras negativas por hematoxilina y eosina (8 %) también fueron negativas con la coloración de Warthin-Starry. Tres de las ocho muestras positivas por esta última, también lo fueron por cultivo. Además, 47 (92,1 %) muestras positivas por hematoxilina y eosina fueron positivas por PCR. Del grupo de 16 negativos por hematoxilina y eosina, 11 (68,8 %) fueron positivos por PCR. De las muestras positivas por PCR, 11 también lo fueron por cultivo (23,4 %). Siete muestras fueron positivas por PCR y Warthin-Starry y tres lo fueron por PCR, Warthin-Starry y cultivo. Todos los fragmentos de la PCR-LipL32 fueron secuenciados y mostraron secuencias específicas de L. interrogans . Conclusiones. Se confirmó la infección por Leptospira y la única lesión presente en el reservorio atribuible fue la nefritis intersticial.

Uropathogenic Escherichia coli causes cortical tubular necrotic cell death and the release of macrophage migration inhibitory factor.

The macrophage migration inhibitory factor (MIF), a pro-inflammatory cytokine, is deregulated in acute kidney injury (AKI) through an unknown mechanism. In the present study, we used a previously described mouse model of ascending urinary tract infection in which uropathogenic Escherichia coli (UPEC) were transurethrally inoculated to induce kidney infections. Here, we show that urinary MIF was upregulated during AKI while MIF was abundantly expressed in the renal cortical tubules and that UPEC infection caused a decrease in tubular MIF. Infections with UPEC in vitro caused MIF release in a cell type-dependent manner, which was independent of receptor-mediated internalization, signal transduction, and transcription. Indeed, UPEC infection-induced necrotic cell death in vitro and in vivo correlated with extracellular acidification and processed MIF secretion. These data suggest that MIF is released by necrotic renal cortical tubular cells during UPEC infection.

Negative regulatory responses to metabolically triggered inflammation impair renal epithelial immunity in diabetes mellitus.

Diabetes mellitus is characterized by chronic inflammation and increased risk of infections, particularly of tissues exposed to the external environment. However, the causal molecular mechanisms that affect immune cells and their functions in diabetes are unclear. Here we show, by transcript and protein analyses, signatures of glucose-induced tissue damage, chronic inflammation, oxidative stress, and dysregulated expression of multiple inflammation- and immunity-related molecules in diabetic kidneys compared with non-diabetic controls. Abnormal signaling involving cytokines, G-protein coupled receptors, protein kinase C isoforms, mitogen-activated protein kinases, nuclear factor-κB (NFκB), and Toll-like receptors (TLR) were evident. These were accompanied by overexpression of negative regulators of NFκB, TLR, and other proinflammatory pathways, e.g., A20, SOCS1, IRAK-M, IκBα, Triad3A, Tollip, SIGIRR, and ST2L. Anti-inflammatory and immunomodulatory molecules, e.g., IL-10, IL-4, and TSLP that favor TH2 responses were strongly induced. These molecular indicators of immune dysfunction led us to detect the cryptic presence of bacteria and human cytomegalovirus in more than one third of kidneys of diabetic subjects but none in non-diabetic kidneys. Similar signaling abnormalities could be induced in primary human renal tubular epithelial (but not mesangial) cell cultures exposed to high glucose, proinflammatory cytokines and methylglyoxal, and were reversed by combined pharmacological treatment with an antioxidant and a PKC inhibitor. Our results suggest that diabetes impairs epithelial immunity as a consequence of chronic and inappropriate activation of counter-regulatory immune responses, which are otherwise physiological protective mechanisms against inflammation. The immune abnormalities and cryptic renal infections described here may contribute to progression of diabetic nephropathy.

Histopathological kidney alterations in rats naturally infected with Leptospira.

INTRODUCTION: Histopathological changes by Leptospira in naturally infected rodent reservoirs have been poorly described. OBJECTIVE: The aim of the current study is to describe renal histopathology associated with leptospirosis infection of naturally infected rodents captured in the urban area of the city of Medellin, Colombia. MATERIALS AND METHODS: We performed hematoxilin-eosin (H-E) on kidney samples collected from 254 captured rodents. The positive samples were processed by Warthin Starry (W-S) staining and PCR-LipL 32. RESULTS: Fifty one rodent kidneys showed H-E histopathological changes that consisted of inflammatory infiltrate with lympho-plasmocitary cells and histiocytes. We performed W-S staining and PCR-LipL 32 to 67 kidney samples, including the 51 that had shown detectable changes by H-E and 16 (8%) of 203 rodents with negative results. Eight of the samples that tested positive for H-E (15.7%) were also positive for W-S staining. All negative for H-E were also negative for W-S staining. Of the W-S positive samples also tested for culture only three tested positive for both. Additionally, 47 (92.1%) samples positive for H-E were positive for PCR; while eleven of the 16 (68.8%) negative for H-E were positive for PCR. The samples positive for PCR were subsequently tested for culture and 11 (23.4%) were positive. Seven samples were positive for PCR and W-S and three were positive for PCR, W-S and culture. All of the PCR-LipL 32 fragments were sequenced and showed specific amplicons for L. interrogans . CONCLUSIONS: The Leptospira infection was confirmed in all of the animals tested. The only histological kidney lesion attributable to leptospiral infection in the reservoir was interstitial nephritis.

[Effect of nanobacteria on cell damage and crystal retention in renal tubular epithelial cells].

OBJECTIVE: To study the damage of nanobacteria on HK-2 cells, the possible principles, the effect of crystals (COM) adhering to HK-2 cells after the damage. METHODS: Four groups were chosen for the study: control group, NB group, nHAP group and COM group. Morphological changes of the HK-2 cells were observed after HE stain and with TEM after 12 hours and 24 hours. Meanwhile, the levels of H2O2, LDH, MDA and ATPases were surveyed after 6 hours,12 hours and 24 hours, respectively. And 6, 12, and 24 hours later, COM crystals were mixed into the culture fluids of each group. Then phalloidin-FITC was used to finish fluorescent staining of the cells. At last, the adhering effects of each group with the laser scanning confocal fluorescence microscope were observed and contrasted. RESULTS: After HE stain and with TEM: in NB and nHAP group, the shape of the cells changed, brush borders were arranged in disorder, vacuoles formed in the kytoplasms, the mitochondria became swelled up, the karyotheca dissolved and the nucleolus disappeared in some cells. After 24 hours, in NB group, the number of the cells in which the karyotheca dissolved was more than that in nHAP group. After 12 and 24 hours, the level of H2O2 in NB group was higher than that in control group and nHAP group; After 6 and 24 hours, the level of MDA in NB group was higher than that in control group and nHAP group; At each time point, there was no significant difference in the level of LDH between control group, nHAP group and NB group; After 12 hours, the activities of Na+/K+ ATPases in NB group and nHAP group were lower than those in control group. And after 24 hours, the activity of Na+/K+ ATPases in NB group was lower than that in control group; After 12 and 24 hours, the activities of Ca2+/Mg2+ ATPases in NB group was lower than those in control group. After 12 hours, the activity of Ca2+/Mg2+ ATPases in nHAP group was lower than that in control group. The observation with the laser scanning confocal fluorescence microscope: after 12 hours, showed that the number of the crystals adhering to the cells in NB group and COM group increased, and in COM group, some crystals had entered the cells; after 24 hours, the adhering effects of the crystals in NB and COM group were similar to those after 12 hours, but the number of adhered crystals was more than that after 12 hours; At each time point, there was no significant change in control and nHAP groups. CONCLUSION: Nanobacteria has a damage effect on HK-2 cells, the damage increases with the acting time expanding. The damage is more severe than that of nHAP. In the damage process of nanobacteria, the lipid peroxidation may play an important role. After the damage of nanobacteria, the adhering effect of the COM crystals to the cells increases observably, and the number of crystals adhering to the cells becomes more and more with the acting time expanding. Although nHAP also has a damage effect on HK-2 cells, it does not effect the adhering process.

Early exposure to germs modifies kidney damage and inflammation after experimental ischemia-reperfusion injury.

Kidney ischemia-reperfusion injury (IRI) is, in part, mediated by immune and inflammatory factors. Since microbial stimuli are known to alter immune and inflammatory responses, we hypothesized that differences in perinatal microbial status would modify renal injury following IRI. We performed bilateral renal IRI on 6-wk-old germ-free and control mice and studied the effects on kidney lymphocyte trafficking, cytokines, function, and structure. Compared with control mice, normal kidneys of germ-free mice exhibited more NKT cells and lower IL-4 levels. Postischemia, more CD8 T cells trafficked into postischemic kidneys of germ-free mice compared with control mice. Renal structural injury and functional decline following IRI were more severe in germ-free mice compared with control mice. When germ-free mice were conventionalized with the addition of bacteria to their diet, the extent of renal injury after IRI became equivalent to age-matched control mice, with similar numbers and phenotypes of T cells and NKT cells, as well as cytokine expression in both normal kidneys and postischemic kidneys of conventionalized germ-free mice and age-matched control mice. Thus microbial stimuli influence the phenotype of renal lymphocytes and the expression of cytokines of normal kidneys and also modulate the outcome of IRI.

Streptozocin-induced diabetic mouse model of urinary tract infection.

Diabetics have a higher incidence of urinary tract infection (UTI), are infected with a broader range of uropathogens, and more commonly develop serious UTI sequelae than nondiabetics. To better study UTI in the diabetic host, we created and characterized a murine model of diabetic UTI using the pancreatic islet beta-cell toxin streptozocin in C3H/HeN, C3H/HeJ, and C57BL/6 mouse backgrounds. Intraperitoneal injections of streptozocin were used to initiate diabetes in healthy mouse backgrounds, as defined by consecutive blood glucose levels of >250 mg/dl. UTIs caused by uropathogenic Escherichia coli (UTI89), Klebsiella pneumoniae (TOP52 1721), and Enterococcus faecalis (0852) were studied, and diabetic mice were found to be considerably more susceptible to infection. All three uropathogens produced significantly higher bladder and kidney titers than buffer-treated controls. Uropathogens did not have as large an advantage in the Toll-like receptor 4-defective C3H/HeJ diabetic mouse, arguing that the dramatic increase in colonization seen in C3H/HeN diabetic mice may partially be due to diabetic-induced defects in innate immunity. Competition experiments demonstrated that E. coli had a significant advantage over K. pneumoniae in the bladders of healthy mice and less of an advantage in diabetic bladders. In the kidneys, K. pneumoniae outcompeted E. coli in healthy mice but in diabetic mice E. coli outcompeted K. pneumoniae and caused severe pyelonephritis. Diabetic kidneys contained renal tubules laden with communities of E. coli UTI89 bacteria within an extracellular-matrix material. Diabetic mice also had glucosuria, which may enhance bacterial replication in the urinary tract. These data support that this murine diabetic UTI model is consistent with known characteristics of human diabetic UTI and can provide a powerful tool for dissecting this infection in the multifactorial setting of diabetes.

Renal magnesium wasting and tubular dysfunction in leptospirosis.

BACKGROUND: Tubulo-interstitial nephritis is the main cause of acute renal injury in leptospirosis. The aim of this study was to evaluate renal tubular function and excretion of solutes in leptospirosis patients during a recent outbreak of leptospirosis in Nan province, Thailand. METHODS: Clinical manifestations were recorded and routine laboratory tests were performed upon admission. Renal tubular functions including tubular reabsorption of phosphate (TRP), fractional excretion of magnesium (FE(Mg)), urinary calcium to creatinine ratio (Uca/cr), urine N-acetyl-beta-D glucosaminidase (NAG) and urine beta(2)-microglobulin were serially monitored during 2 weeks after admission. RESULTS: A total of 20 leptospirosis patients were recruited. Nine (45%) patients had acute renal failure (ARF). Increased urine NAG and beta(2)-microglobulin, which indicate proximal tubular dysfunction, were demonstrated in all 20 (100%) patients. Fifteen (75%) patients had hypermagnesuria, whereas 10 (50%) patients had decreased TRP. Renal magnesium (Mg) and phosphate (P) wasting caused hypomagnesaemia and hypophosphataemia in nine and three patients with ARF, respectively. These abnormal findings significantly improved within 2 weeks after admission. CONCLUSIONS: We conclude that renal Mg and P wasting commonly occur in patients with leptospirosis. The measurement of Mg and P levels in both serum and urine of leptospirosis patients, especially those with ARF, is therefore highly recommended.

[Adaptation of an immunohistochemistry protocol for the detection of Leptospira spp. in samples of formaldehyde-fixed tissue]. / Adaptación de un protocolo de inmunohistoquímica para la detección de Leptospira spp. en muestras de tejido fijado en formaldehído.

The immunohistochemistry technique was evaluated in tissue samples fixed in formaldehyde saline solution 10 % and included in paraffin to be used as a lab method allowing to identify leptospires in tissues. Samples obtained from the experimental inoculation of 8 guinea pigs carriers of L. interrogans Pomona isolated from a clinical case were used. The disease was reproduced in a lab model. The histologic sections of the kidneys of the animals inoculated were subjected to histopathological studies, immunofluorescence, Warthin-Starry stain, and immunohistochemistry technique using formaldehyde-fixed samples. This technique proved to be an efficient tool for the diagnosis of leptospirosis.

Urothelial CD44 facilitates Escherichia coli infection of the murine urinary tract.

Escherichia coli is the most common pathogen found in urinary tract infections (UTIs), mainly affecting children and women. We report that CD44, a hyaluronic acid (HA) binding protein that mediates cell-cell and cell-matrix interactions, facilitates the interaction of E. coli with urothelial cells and thus the infection of the host. We found that CD44 is constitutively expressed on urothelial cells and that HA accumulates in E. coli-induced UTI. In CD44-deficient mice, the bacterial outgrowth was dramatically less compared with wild-type mice despite similar granulocyte influx in the bladder and in the kidney as well as comparable cytokines/chemokines levels in both genotypes. E. coli was able to bind HA, which adhered to CD44-positive tubular epithelial cells. Most importantly, the interaction of CD44 on tubular epithelial cells with HA facilitated the migration of E. coli through the epithelial monolayer. The results provide evidence that CD44 on urothelial cells facilitates E. coli UTI. Disruption of the interaction between CD44 and HA in the bladder may provide a new approach to prevent and to treat UTI.

Interleukin-8 secretion of cortical tubular epithelial cells is directed to the basolateral environment and is not enhanced by apical exposure to Escherichia coli.

In upper urinary tract infections, tubular epithelial cells (TEC) may play a pivotal role in the initiation of the renal inflammatory response. They exert crucial immunological functions such as processing and presentation of foreign antigen, secretion of proinflammatory cytokines (interleukin-6 [IL-6] and tumor necrosis factor alpha) and chemokines (IL-8, MCP-1, ENA-78, and RANTES). Since monolayer cultures are a limited model for polarized tubular epithelial cells, we studied the side-dependent IL-8 secretion of TEC by using cell culture inserts as a basement membrane imitation. Primary cultures of proximal TEC were stimulated with differently fimbriated mutants of Escherichia coli, E. coli LPS, S-fimbria isolates, and IL-1alpha. IL-8 protein was measured by enzyme-linked immunosorbent assay, and IL-8-like biological activity was tested by measuring elastase release from polymorphonuclear cells in supernatants of the upper and lower compartments. IL-8 mRNA was compared by competitive PCR. IL-8 secretion by TEC into the basolateral environment was significantly higher than secretion into the apical compartment, representing the tubular lumen. However, stimulation of IL-8 secretion by TEC was restricted to IL-1alpha and was not inducible by E. coli mutants, S fimbriae, or lipopolysaccharide. With this in vitro model of polarized TEC, we show that luminal contact of TEC with uropathogenic E. coli does not result in enhanced IL-8 secretion. The basolaterally directed production of the neutrophil chemotactic factor IL-8 by TEC after stimulation with IL-1alpha might play an important role in the initiation of inflammatory cell influx into the renal parenchyma.