mTORC2 critically regulates renal potassium handling

The mTOR pathway orchestrates cellular homeostasis. The rapamycin-sensitive mTOR complex (mTORC1) in the kidney has been widely studied; however, mTORC2 function in renal tubules is poorly characterized. Here, we generated mice lacking mTORC2 in the distal tubule (Rictor^fl/fl Ksp-Cre mice), which were viable and had no obvious phenotype, except for a 2.5-fold increase in plasma aldosterone. Challenged with a low-Na⁺ diet, these mice adequately reduced Na⁺ excretion; however, Rictor^fl/fl Ksp-Cre mice rapidly developed hyperkalemia on a high-K⁺ diet, despite a 10-fold increase in serum aldosterone levels, implying that mTORC2 regulates kaliuresis. Phosphorylation of serum- and glucocorticoid-inducible kinase 1 (SGK1) and PKC-α was absent in Rictor^fl/fl Ksp-Cre mice, indicating a functional block in K⁺ secretion activation via ROMK channels. Indeed, patch-clamp experiments on split-open tubular segments from the transition zone of the late connecting tubule and early cortical collecting duct demonstrated that Ba²⁺-sensitive apical K⁺ currents were barely detectable in the majority of Rictor^fl/fl Ksp-Cre mice. Conversely, epithelial sodium channel (ENaC) activity was largely preserved, suggesting that the reduced ability to maintain K⁺ homeostasis is the result of impaired apical K⁺ conductance and not a reduced electrical driving force for K⁺ secretion. Thus, these data unravel a vital and nonredundant role of mTORC2 for distal tubular K⁺ handling.     

Ciliary calcium signaling is modulated by kidney injury molecule-1 (Kim1)

Primary cilia have been shown to play an important role in embryonic development as well as in postnatal life. Dysfunctional cilia are associated with situs inversus, retinal abnormalities, impaired mucociliary clearance, infertility, hydrocephalus, and congenital renal cysts. In autosomal dominant polycystic kidney disease, mutations of the ciliary proteins polycystin1 or the transient receptor potential (TRP) channel family protein polycystin2 (TRPP2) cause progressive cyst formation and destruction of the kidney. Primary cilia act as flow sensors and respond to flow-mediated bending with a prolonged intracellular calcium increase, which appears to require an intact polycystin protein complex. We have established a novel flow chamber system, which allows us to study renal epithelial cells by live cell imaging. We show that MDCK cells respond to flow by a delayed increase in intracellular calcium and that this response requires these cells to be ciliated. We show that a novel interactor of TRPP2, kidney injury molecule-1 (Kim1), which is expressed at low levels in the normal kidney and upregulated after ischemia, in renal cell cancer and in PKD is targeted to primary cilia when stably expressed in MDCK cells. We demonstrate that expression of tyrosine mutant Kim1, lacking a conserved tyrosine in the intracellular tail, abolishes the calcium increase in response to flow in a dominant negative manner. These results establish Kim1 as a novel regulatory molecule of flow-induced calcium signaling. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

A comprehensive survey of pediatric diarrhea at a private hospital in Metro Manila.

The epidemiology of diarrhea among Filipino pediatric patients, representing a cross-section of socioeconomic strata, was investigated over a one year period. Rotavirus was detected in 33.9% of the diarrhea stools examined and was the leading cause of diarrhea in the study population. Although proportionately more rotavirus was found during the cold season, most children became infected with rotavirus during the rainy season, when diarrheal disease was at its peak in Metropolitan Manila. Enteric adenovirus types 40 or 41 were associated with only 5.4% of the diarrhea cases. Overall, one or more etiologic agents of diarrhea were detected in 67.2% of the stools examined. Many of these positive stools (21.6%) contained multiple diarrheogenic agents. Bacterial enteric pathogens were isolated from 32.3% of the cases. Nearly 70% of these patients with bacterial gastroenteritis became ill during the rainy season. Etiology specific and general risk factors associated with diarrheal illness in the study population are discussed.     

Short report: High incidence of shigellosis among Peruvian soldiers deployed in the Amazon River basin

We investigated the etiology of acute diarrhea among Peruvian military recruits undergoing three months of basic combat training near the Amazonian city of Iquitos. From January through September 2002, 307 of 967 recruits were seen at the Health Post for diarrhea (attack rate [AR] = 31.8%, incidence = 1.28 95% confidence interval [CI] = 1.14-1.43] episodes/person-year). Shigella spp. were the most common bacterial pathogen recovered from recruits experiencing diarrhea episodes. These bacteria were isolated from 89 (40%) of 225 diarrheal stools examined (AR = 7.6%, incidence = 0.30 [95% CI = 0.24-0.38] episodes/person-year). Most (83 of 90; 92%) of the Shigella isolates were S. flexneri, of which 57 (69%) were serotype 2a. Seventy-six percent of Shigella isolates were resistant to sulfamethoxazole/trimethoprim and all were sensitive to ciprofloxacin. Peruvian soldiers may be an excellent population in which to test the efficacy of S. flexneri vaccines in advanced development.     

Preparation and successful engraftment of purified CD34+ bone marrow progenitor cells in patients with non-Hodgkin's lymphoma

From September 1992 to January 1994, we evaluated the use of the CEPRATE SC stem cell concentrator (CellPro, Inc, Bothell, WA) to select CD34+ cells from the bone marrow (BM) of 25 patients with non-Hodgkin's lymphoma in complete remission. This system uses the biotinylated 12.8 IgM MoAb to select CD34+ cells. Cells are retained on an avidin column and detached by agitation. Fifteen patients have been transplanted with the CD34+ purified fraction. The CD34+ purified fraction of the 25 processed BMs contained a median of 0.54% of the original nucleated cells in a volume of 5 to 10 mL. The median concentration of CD34+ cells was 49% (range, 12% to 80%), and the median enrichment of CD34+ cells was 33-fold (range, 9- to 85-fold). This selected CD34+ fraction retained 60% (range, 15% to 95%) of late granulocyte-macrophage colony- forming units (CFU-GM), 55% (range, 12% to 99%) of early CFU-GM, and 31% (range, 2% to 100%) erythroid burst-forming units (BFU-E) corresponding to median enrichments of 22-fold (range, 1- to 71-fold), 19-fold (range, 2- to 58-fold), and 14-fold (range, 2- to 200-fold), respectively. There was a correlation between immune phenotypes and progenitor cells. In the initial buffy-coat fractions, the percentage of CD34+ cells was correlated to the cloning efficiency of both late CFU-GM (P < .05) and early CFU-GM (P < .001). In the final selected fraction, there was a correlation between the percentage of CD34+/CD33- and the cloning efficiency of early CFU-GM (P < .05) and between the percentage of CD34+/CD33+ and the cloning efficiency of late CFU-GM (P < .05). Lymphoma cells positive for t(14; 18) were found by polymerase chain reaction in 9 of 14 buffy coats tested before CD34+ cell purification. In 8 cases, the CD34(+)-selected fraction was found to be negative, and the CD34- fraction was found to be positive. After cryopreservation, the recoveries of progenitor cells in the CD34(+)- purified fraction were 79% for late CFU-GM, 71% for early CFU-GM, and 73% for BFU-E. The 15 patients transplanted with the concentrated CD34+ fraction received a median dose of 1 x 10(6) CD34+ cells/kg (range, 0.3 to 2.96) and 10.62 x 10(4) early CFU-GM/kg (range, 0.92 to 25.55). Median days to recovery to 0.5 x 10(9)/L neutrophils and 50 x 10(9)/L platelets were days 15 (range, 10 to 33) and 23 (range, 11 to 68), respectively.(ABSTRACT TRUNCATED AT 400 WORDS)     

Recombinant human MCP-1/JE induces chemotaxis, calcium flux, and the respiratory burst in human monocytes

The JE gene was first described as a platelet-derived growth factor (PDGF)-inducible gene in mouse 3T3 cells. The human homologue of JE encodes a protein whose predicted amino acid sequence is identical to that of the monocyte chemoattractant MCP-1 (also called MCAF and SMC-CF), which belongs to a recently identified family of small secretory proteins with cytokine properties. We purified recombinant human MCP-1/JE (hMCP-1/JE) produced in COS cells and demonstrated that it is chemotactic for human monocytes with a specific activity similar to natural MCP-1. In addition, pure recombinant hMCP-1/JE stimulates monocytes, inducing an increase in cytosolic free calcium and the respiratory burst, but is completely inactive on human neutrophils. These results help to define functionally a well-known growth factor-inducible gene and a member of a new family of cytokines.     

Amino acid sequence of human prothrombin fragments 1 and 2.

The amino acid sequence of the nonthrombin half of human prothrombin is presented. Prothrombin fragment 1 has 155 amino acid residues as compared with 156 for the bovine equivalent. Ten gamma-carboxyglutamic acid residues are at the same location in each species. Human prothrombin fragment 2 contains 118 amino acid residues, as does the similar bovine fragment. Comparing bovine and human prothrombin fragment 1 we found 131 residues to be identical (84%). In prothrombin fragment 2, 84 residues were identical (71%). Assuming a time span of 90 million years since the radiation of several orders of placental mammals, prothrombin fragments 1 and 2 incorporated one substitution site per 100 amino acid sites every 11.2 and 6.3 million years, respectively. Internal homology is acribed to partial gene duplication, with the most likely crossover point located between residues 60-61 and residues 165-166.     

Amifostine improves the antileukemic therapeutic index of mafosfamide: implications for bone marrow purging

One of the principal challenges of cancer chemotherapy is the relative inability of most anticancer drugs to distinguish between normal and neoplastic tissues. Consequently, a broad range of toxicities are experienced by patients, especially myelosuppression. Amifostine, a phosphorylated aminothiol, increases the selectivity of specific anticancer drugs for neoplastic cells by protecting normal tissues. One potential application of this protector is during bone marrow purging to selectively remove contaminating cancer cells. This study took normal or leukemic marrow from human subjects and evaluated the ability of amifostine to selectively protect normal bone marrow progenitor cells versus leukemic progenitor cells from the cytotoxic effect of mafosfamide. The dose response of mafosfamide amifostine on leukemia colony-forming units or normal marrow progenitor cells was determined and the LD95 was calculated. Amifostine pretreatment resulted in a statistically significant protection of granulocyte-macrophage colony- forming units and erythroid blast-forming units from the toxicity of mafosfamide (P = .031). Thus, amifostine protection of normal marrow progenitor cells allows a higher LD95 concentration of mafosfamide to be used in ex vivo purging. In contrast, amifostine pretreatment increased the cytotoxicity of mafosfamide on the fresh human leukemia progenitor cells (P = .006). The dual effect of amifostine protection of normal marrow progenitor cells coupled with amifostine-induced sensitization of the leukemia cells increases the possible cell-kill of leukemic stem cells. With amifostine pretreatment, at the LD95 concentrations of mafosfamide for marrow progenitor cells, there was an estimated 6 log increase in cell-kill of the leukemia cells. This selective cell-kill offers the potential for lowering the incidence of leukemic relapse, while preserving more normal stem cells for autologous transplantation.