Miguel A. Lanaspa

Uric acid-induced endothelial dysfunction is associated with mitochondrial alterations and decreased intracellular ATP concentrations

Background/Aims: Endothelial dysfunction is associated with mitochondrial alterations. We hypothesized that uric acid (UA), which can induce endothelial dysfunction in vitro and in vivo, might also alter mitochondrial function. Methods: Human aortic endothelial cells were exposed to soluble UA and measurements of oxidative stress, nitric oxide, mitochondrial density, ATP production, aconitase-2 and enoyl Co-A hydratase-1 expressions, and aconitase-2 activity in isolated mitochondria were determined. The effect of hyperuricemia induced by uricase inhibition in rats on renal mitochondrial integrity was also assessed. Results: UA-induced endothelial dysfunction was associated with reduced mitochondrial mass and ATP production. UA also decreased aconitase-2 activity and lowered enoyl CoA hydratase-1 expression. Hyperuricemic rats showed increased mitDNA damage in association with higher levels of intrarenal UA and oxidative stress. Conclusions: UA-induced endothelial dysfunction is associated with mitochondrial alterations and decreased intracellular ATP. These studies provide additional evidence for a deleterious effect of UA on vascular function that could be important in the pathogenesis of hypertension and vascular disease.

Molecular mechanisms of angiotensin II stimulation on aquaporin-2 expression and trafficking.

ANG II plays a major role in renal water and sodium regulation. In the immortalized mouse renal collecting duct principal cells (mpkCCD(cl4)) cell line, we treated cells with ANG II and examined aquaporin-2 (AQP2) protein expression, trafficking, and mRNA levels, by immunoblotting, immunofluorescence, and RT-PCR. After 24-h incubation, ANG II-induced AQP2 protein expression was observed at the concentration of 10(-10) M and increased in a dose-dependent manner. ANG II (10(-7) M) increased AQP2 protein expression and mRNA levels at 0.5, 1, 2, 6, and 24 h. Immunofluorescence studies showed that ANG II increased the apical membrane targeting of AQP2 from 30 min to 6 h. Next, the signaling pathways underlying the ANG II-induced AQP2 expression were investigated. The PKC inhibitor Ro 31-8220 (5 × 10(-6) M) and the PKA inhibitor H89 (10(-5) M) blocked ANG II-induced AQP2 expression, respectively. Calmodulin inhibitor W-7 markedly reduced ANG II- and/or dDAVP-stimulated AQP2 expression. ANG II (10(-9) M) and/or dDAVP (10(-10) M) stimulated AQP2 protein levels and cAMP accumulation, which was completely blocked by pretreatment with the vasopressin V2 receptor (V2R) antagonist SR121463B (10(-8) M). Pretreatment with the angiotensin AT(1) receptor (AT1R) antagonist losartan (3 × 10(-6) M) blocked ANG II (10(-9) M)-stimulated AQP2 protein expression and cAMP accumulation, and partially blocked dDAVP (10(-10) M)- and dDAVP+ANG II-induced AQP2 protein expression and cAMP accumulation. In conclusion, ANG II regulates AQP2 protein, trafficking, and gene expression in renal collecting duct principal cells. ANG II-induced AQP2 expression involves cAMP, PKC, PKA, and calmodulin signaling pathways via V2 and AT(1) receptors.

Autoimmunity in the pathogenesis of hypertension

Hypertension affects more than one-third of the adult population of the world. However, the cause of high blood pressure is unknown in the vast majority of patients, classified as patients with essential hypertension. Evidence accumulated over the past decade supports the participation of inflammation in the development of experimental hypertension. Investigations have also demonstrated that immune reactivity to overexpressed heat shock protein 70 (HSP70) is involved in the pathogenesis of salt-induced hypertension. This article reviews, first, the role of T cell-induced inflammation in the arteries, kidney and central nervous system in hypertension and the amelioration of hypertension induced by regulatory T cells. Second, experiments showing that autoimmunity directed to HSP70 in the kidney impairs the pressure natriuresis relationship and has a pivotal role in the pathogenesis of salt sensitive hypertension. Finally, we highlight the clinical evidence that supports the participation of autoimmunity in essential hypertension.

The tight junction protein, MUPP1, is up-regulated by hypertonicity and is important in the osmotic stress response in kidney cells

Antibody array proteomics was used to detect differentially expressed proteins in inner medullary collecting duct 3 (IMCD3) cells grown under isotonic and chronic hypertonic conditions. Of 512 potential proteins, >90% were unchanged in expression. Noteworthy was the up-regulation of several tight junction-related proteins, including MUPP1 (multi-PDZ protein-1), ZO1 (zonula occludens 1), and Af6. The most robustly up-regulated protein under hypertonic conditions was MUPP1 (7.2×, P < 0.001). Changes in expression for MUPP1 were verified by quantitative PCR for message and Western blot for protein. In mouse kidney tissues, MUPP1 expression was substantial in the papilla and was absent in the cortex. Furthermore, MUPP1 expression increased 253% (P < 0.01) in the papilla upon 36 h of thirsting. Localization of MUPP1 protein expression was confirmed by immunocytochemical analysis demonstrating only minor staining under isotonic conditions and the substantial presence in chronically adapted cells at the basolateral membrane. Message and protein half-life in IMCD3 cells were 26.2 and 17.8 h, respectively. Osmotic initiators of MUPP1 expression included NaCl, sucrose, mannitol, sodium acetate, and choline chloride but not urea. Stable IMCD3 clones silenced for MUPP1 expression used the pSM2-MUPP1 vector. In cell viability experiments, clones silenced for MUPP1 demonstrated only a minor loss in cell survival under acute sublethal osmotic stress compared with empty vector control cells. In contrast, a 24% loss (P < 0.02) in transepithelial resistance for monolayers of MUPP1-silenced cells was determined as compared with controls. These results suggest that MUPP1 specifically, and potentially tight junction complexes in general, are important in the renal osmoadaptive response.

The expression of aquaporin-1 in the medulla of the kidney is dependent on the transcription factor associated with hypertonicity, TonEBP

Expression of aquaporin-1 (AQP1) and -2 (AQP2) channels in the kidney are critical for the maintenance of water homeostasis and the operation of the urinary concentrating mechanism. Hypertonic stress induced in inner medullary (IMCD3) cells by addition of NaCl to the medium substantially up-regulated the mRNA and protein expression of AQP1, suggesting that its activation occurs at a transcriptional and a translational levels. In contrast, no up-regulation of AQP1 was observed when these cells were exposed to the same tonicity by addition of urea. To explore the transcriptional activation of aqp1 under hypertonic stress, we examined the role of the transcription factor associated with hypertonicity, TonEBP. Treatment of IMCD3 cells with the TonEBP inhibitor rottlerin or silencing its expression with specific shRNA technology led to a substantial reduction in AQP1 expression under hypertonic conditions. Moreover, we defined a conserved TonEBP binding site located 811 bp upstream of the aqp1 exon that is essential for its expression. Single site-directed mutation of this TonE site led to a 54 ± 5% (p < 0.01) decrease in AQP1 luciferase-driven activity under hypertonic stress. TonEBP mutant mice display marked decrement in the expression of AQP1 in the inner medulla. In conclusion, these data demonstrate that TonEBP is necessary for the regulation of AQP1 expression in the inner medulla of the kidney under hypertonic conditions.

Hypertonic stress increases claudin-4 expression and tight junction integrity in association with MUPP1 in IMCD3 cells

We reported that the multiple PDZ protein 1 (MUPP1) is an osmotic response protein in kidney cells. This up-regulation was found to be necessary for the maintenance of tight epithelial properties in these cells. We investigated whether an interaction with one or more members of the claudin family is responsible for this observation. In response to hypertonicity, the up-regulation of claudin-4 (Cldn4) expression, and not other claudins, was initially identified in inner medullary collecting duct (IMCD3) cells by gene array and further verified by quantitative PCR and Western blotting. In kidney tissues, Cldn4 expression was substantial in the papilla and absent in the cortex. Furthermore, Cldn4 expression significantly increased in the papilla of mice after 36 h of thirsting. Cldn4 immunofluorescence in hypertonically stressed cells revealed colocalization with MUPP1 at the tight junctions. Interaction between Cldn4 and MUPP1 was also demonstrated by coimmunoprecipitation of both proteins from IMCD3 cells chronically adapted to hypertonicity. In IMCD3 cells stably silenced for MUPP1 expression under hypertonic conditions, a significant decrement in Cldn4 expression was observed that was restored after inhibition of lysosome activity. Immunofluorescence detection identified that in these MUPP1-silenced cells Cldn4 was mistargeted to the lysosomes. Functionally, silencing Cldn4 expression in IMCD3 cells resulted in a decrease in the transepithelial resistance to the same degree as observed when MUPP1 expression was silenced, suggesting that MUPP1 contributes to the maintenance of a tight epithelium in the medulla of the kidney under hypertonic stress by correctly localizing Cldn4 to the tight junctions.

Inadequate Efficacy of a New Formulation of Fosmidomycin-Clindamycin Combination in Mozambican Children Less than Three Years Old with Uncomplicated Plasmodium falciparum Malaria

ABSTRACT The combination of fosmidomycin and clindamycin (F/C) is effective in adults and older children for the treatment of malaria and could be an important alternative to existing artemisinin-based combinations (ACTs) if proven to work in younger children. We conducted an open-label clinical trial to assess the efficacy, safety, and tolerability of F/C for the treatment of uncomplicated P. falciparum malaria in Mozambican children <3 years of age. Aqueous solutions of the drugs were given for 3 days, and the children were followed up for 28 days. The primary outcome was the PCR-corrected adequate clinical and parasitological response at day 28. Secondary outcomes included day 7 and 28 uncorrected cure rates and fever (FCT) and parasite (PCT) clearance times. Fifty-two children were recruited, but only 37 patients were evaluable for the primary outcome. Day 7 cure rates were high (94.6%; 35/37), but the day 28 PCR-corrected cure rate was 45.9% (17/37). The FCT was short (median, 12 h), but the PCT was longer (median, 72 h) than in previous studies. Tolerability was good, and most common adverse events were related to the recurrence of malaria. The poor efficacy observed for the F/C combination may be a consequence of the new formulations used, differential bioavailability in younger children, naturally occurring variations in parasite sensitivity to the drugs, or an insufficient enhancement of their effects by naturally acquired immunity in young children. Additional studies should be conducted to respond to the many uncertainties arising from this trial, which should not discourage further evaluation of this promising combination.

Asymptomatic Hyperuricemia Without Comorbidities Predicts Cardiometabolic Diseases: Five-Year Japanese Cohort Study

Whether asymptomatic hyperuricemia in the absence of comorbidities increases the risk for cardiometabolic disorders and chronic kidney disease remains controversial. This study was conducted to clarify the association between asymptomatic hyperuricemia and cardiometabolic conditions. Subjects consisting of Japanese adults between 30 and 85 years of age were enrolled in the study at Center for Preventive Medicine, St Luke’s International Hospital, Tokyo, and were available at enrollment (2004) and at 5-year follow-up (2009). Subjects were excluded if they were overweight or obese, hypertensive, diabetic, and dyslipidemic, had a history of gout or hyperuricemia on medications, or had chronic kidney disease as estimated glomerular filtration rate <60 mL/min per 1.73 m2. Linear and logistic regression analyses were used to examine the relationship between hyperuricemia and development of hypertension, diabetes mellitus, dyslipidemia, chronic kidney disease, and overweight/obesity (unadjusted and adjusted for age, sex, smoking, drinking habits, baseline estimated glomerular filtration rate, and body mass index). Five thousand eight hundred and ninety-nine subjects without comorbidities (mean age of 47±10 years, 1864 men) were followed for 5 years. Hyperuricemia (defined as >7 mg/dL in men and ≥6 mg/dL in women) was associated with increased cumulative incidence of hypertension (14.9% versus 6.1%; P<0.001), dyslipidemia (23.1% versus 15.5%; P<0.001), chronic kidney disease (19.0% versus 10.7%; P<0.001), and overweight/obesity (8.9% versus 3.0%; P<0.001), while diabetes mellitus (1.7% versus 0.9%; P=0.087) showed a trend but did not reach statistical significance. In conclusion, asymptomatic hyperuricemia carries a significant risk for developing cardiometabolic conditions in Japanese individual without comorbidities.

Elevated Serum Uric Acid Level Predicts Rapid Decline in Kidney Function

Background: While elevated serum uric acid level (SUA) is a recognized risk factor for chronic kidney disease, it remains unclear whether change in SUA is independently associated with change in estimated glomerular filtration rate (eGFR) over time. Accordingly, we examined the longitudinal associations between change in SUA and change in eGFR over 5 years in a general Japanese population. Methods: This was a large, single-center, retrospective 5-year cohort study at St. Lukes International Hospital, Tokyo, Japan, between 2004 and 2009. We included 13,070 subjects (30-85 years) in our analyses whose data were available between 2004 and 2009. Of those, we excluded 492 subjects with eGFR <60 mL/min/1.73 m2 at baseline. In addition to examining the entire cohort (n = 12,578), we stratified our analyses by baseline eGFR groups: 60-90, 90-120, and ≥120 mL/min/1.73 m2. Linear and logistic regressions models were applied to examine the relationships between baseline and change in SUA, change in eGFR, and rapid eGFR decline (defined as the highest quartile of change in eGFR), adjusted for age, gender, body mass index, abdominal circumference, hypertension, dyslipidemia, and diabetes mellitus. Results: After multivariable adjustments including baseline eGFR, 1 mg/dL increase in baseline SUA was associated with greater odds of developing rapid eGFR decline (OR 1.27, 95% CI 1.17-1.38), and 1 mg/dL increase in SUA over 5 years was associated with 3.77-fold greater odds of rapid eGFR decline (OR 3.77, 95% CI 3.35-4.26). Conclusions: Elevated baseline SUA and increasing SUA over time were independent risk factors for rapid eGFR decline over 5 years.

Detection of Streptococcus pneumoniae and Haemophilus influenzae type B by real-time PCR from dried blood spot samples among children with pneumonia: a useful approach for developing countries.

Background Dried blood spot (DBS) is a reliable blood collection method for storing samples at room temperature and easily transporting them. We have previously validated a Real-Time PCR for detection of Streptococcus pneumoniae in DBS. The objective of this study was to apply this methodology for the diagnosis of S. pneumoniae and Haemophilus influenzae b (Hib) in DBS samples of children with pneumonia admitted to two hospitals in Mozambique and Morocco. Methods Ply and wzg genes of S. pneumoniae and bexA gene of Hib, were used as targets of Real-Time PCR. 329 DBS samples of children hospitalized with clinical diagnosis of pneumonia were tested. Results Real-Time PCR in DBS allowed for a significant increase in microbiological diagnosis of S. pneumoniae and Hib. When performing blood bacterial culture, only ten isolates of S. pneumoniae and none of Hib were detected (3·0% positivity rate, IC95% 1·4-5·5%). Real-Time PCR from DBS samples increased the detection yield by 4x fold, as 30 S. pneumoniae and 11 Hib cases were detected (12·4% positivity rate, IC95% 9·0-16·5%; P<0·001). Conclusion Real-Time PCR applied in DBS may be a valuable tool for improving diagnosis and surveillance of pneumonia caused by S. pneumoniae or Hib in developing countries.