Tubular expression of heat-shock protein 27 inhibits fibrogenesis in obstructive nephropathy.

Morphological changes that occur during kidney injury involve actin skeleton remodeling. Here we tested whether heat-shock protein 27 (HSP27), a small stress response protein involved in cytoskeletal remodeling, protects the kidney from tubulointerstitial fibrosis in obstructive nephropathy. Tubular cell HSP27 immunostaining was significantly increased in human kidneys with ureteropelvic junction obstruction, supporting the clinical relevance of our studies. To develop an animal model for mechanistic studies, we generated transgenic mice that specifically overexpress human HSP27 in renal tubules, under the kidney androgen-regulated protein promoter, and determined the effects of HSP27 overexpression on epithelial-to-mesenchymal transition and tubulointerstitial fibrosis following unilateral ureteral obstruction. This was associated with decreased fibrogenesis as evidenced by significant declines in phosphorylated p38MAPK, collagen III, α-smooth muscle actin, 4-hydroxynonenal, and reduced trichrome staining following obstruction. Notably, E-cadherin and ß-catenin remained at the cell membrane of tubular cells in transgenic mice with an obstructed ureter. Monocyte/macrophage infiltration, however, was not significantly affected in these transgenic mice. Thus, tubular HSP27 inhibits fibrogenesis in obstructive nephropathy. Further studies are needed to determine pathways regulating the interactions between HSP27 and the E-cadherin-ß-catenin complex.

WITHDRAWN: Endothelium in the allograft.

The paper entitled "Endothelium in the allograft" by Bryan N Becker et al, which was published online on 9 September 2009, has been withdrawn at the authors' request. Kidney International advance online publication, 9 September 2009; doi:10.1038/ki.2009.333.

Local television news reporting of kidney disease.

BACKGROUND: Local television is the primary news source for the majority of Americans. This study aims to describe how local news reports on kidney disease. METHODS: Using our searchable database of health-related late local news segments from 2002, we identified stories with the key words kidney, hypertension, blood pressure, or diabetes. This database is a representative sample of the late local news on 122 stations in the 50 largest US media markets, comprising 60% of the population. The content of each identified story was reviewed to determine whether it mentioned: (1) chronic kidney disease (CKD), (2) screening for kidney disease, or (3) kidney disease as a potential complication (for blood pressure- or diabetes-related stories). RESULTS: Only 2 of 1,799 database news stories (0.11%) included "kidney" as a summary key word; neither referred to CKD, screening, or complications of other diseases. Of 19 stories about hypertension or blood pressure (1.06% of all stories) and the 14 stories about diabetes (0.78% of all stories), none mentioned these criteria. CONCLUSION: Despite efforts to increase public awareness of and screening for CKD, local television news (the most important news source for a majority of Americans) did little to help achieve these goals. Further work will be needed to confirm whether this paucity of coverage varies over time and determine why so little attention is given to CKD. Educating physicians and public relations personnel who advocate for kidney disease about journalists' needs may be an important step to help advance public awareness of CKD.

Pancreatic precursors and differentiated islet cell types from murine embryonic stem cells: an in vitro model to study islet differentiation.

Embryonic stem (ES) cells differentiating in vitro reproduce many facets of early embryonic development, including the expression of developmentally regulated transcription factors and the differentiation of multipotent precursor cells. ES cells were evaluated for their ability to differentiate into pancreatic and islet lineage-restricted stages including pancreatic duodenal homeobox 1 (PDX1)-positive pancreatic precursor cells, early endocrine cell progenitors, and islet hormone-producing cells. Following growth and differentiation in nonselective medium containing serum, murine ES cells spontaneously differentiated into cells individually expressing each of the four major islet hormones: insulin, glucagon, somatostatin, and pancreatic polypeptide. PDX1 immunostaining cells appeared first, before hormone-positive cells had emerged. Hormone-positive cells appeared within focal clusters of cells coexpressing PDX1 and the nonclassical hormone markers peptide YY (YY) and islet amyloid polypeptide (IAPP) in combination with the definitive hormones, characteristic of endocrine cells appearing during early pancreaticogenesis. This system allows the investigation of many facets of islet development since it promotes the appearance of the complete range of islet phenotypes and reproduces important developmental stages of normal islet cytodifferentiation in differentiating ES cell cultures.

Chemokines and their receptors in human renal allotransplantation.

BACKGROUND: Chemokines and their receptors play a critical role in leukocyte trafficking, and inhibition of select chemokines has been shown to attenuate kidney disease and allograft rejection in animal models. Therefore, we evaluated chemokine and chemokine receptor transcripts in human renal allograft biopsies, correlating transcript levels with clinical course and immunohistochemical analysis to relate chemokine expression to relevant clinical human disease phenotypes. METHODS: Renal biopsies were grouped as postreperfusion (n=10), stable function (n=10), subclinical (n=10) or acute rejection (n=17), or calcineurin inhibitor nephrotoxicity (n=9) based on clinical presentation and histopathologic assessment. Using quantitative real-time polymerase chain reaction analysis, chemokine transcripts were assessed relative to transcript levels in preprocurement biopsies from live donor kidneys (n=15). RESULTS: Transcripts from several inflammatory chemokines (CCL3, CCL5, CXCL9, CXCL10, and CXCL11) and chemokine receptors (CCR5, CCR7, and CXCR3) were significantly increased in allografts with subclinical and clinical acute rejection, indicating a strong polarization toward a T-helper 1 effector phenotype during rejection. These transcripts also distinguished acutely rejecting allografts from allografts with nonrejection causes of renal dysfunction. Biopsies from patients with stable function without histologic evidence of rejection had increased chemokine transcript levels that were qualitatively similar but quantitatively reduced compared with rejecting allografts. CONCLUSIONS: This comprehensive evaluation of chemokines and their receptors in human renal transplantation defines associations between chemokine expression and clinical phenotypes, may have diagnostic utility, and highlights relevant pathways for therapeutic intervention.

Functionally significant renal allograft rejection is defined by transcriptional criteria.

Renal allograft acute cellular rejection (ACR) is a T-cell mediated disease that is diagnosed histologically. However, many normally functioning allografts have T-cell infiltrates and histological ACR, and many nonimmune processes cause allograft dysfunction. Thus, neither histological nor functional criteria are sufficient to establish a significant rejection, and the fundamental features of clinical rejection remain undefined. To differentiate allograft lymphocyte infiltration from clinically significant ACR, we compared renal biopsies from patients with ACR to patients with: sub-clinical rejection (SCR, stable function with histological rejection); no rejection; and nontransplanted kidneys. Biopsies were compared histologically and transcriptionally by RT-PCR for 72 relevant immune function genes. Neither the degree nor the composition of the infiltrate defined ACR. However, transcripts up-regulated during effector T(H)1 T-cell activation, most significantly the transcription factor T-bet, the effector receptor Fas ligand and the costimulation molecule CD152 clearly (p = 0.001) distinguished the patient categories. Transcripts from other genes were equivalently elevated in SCR and ACR, indicating their association with infiltration, not dysfunction. Clinically significant ACR is not defined solely by the magnitude nor composition of the infiltrate, but rather by the transcriptional activity of the infiltrating cells. Quantitative analysis of selected gene transcripts may enhance the clinical assessment of allografts.

ATR blockade reduces IFN-gamma production in lymphocytes in vivo and in vitro.

BACKGROUND: Type 1 angiotensin II (Ang II) receptor (AT(1)R) signaling induces proinflammatory responses. Recent studies suggest that T lymphocytes express AT(1)R; yet the effects of Ang II binding to AT(1)R on T cells are poorly understood. We examined the effect of AT(1)R blockade on release of the proinflammatory cytokine, interferon-gamma (IFN-gamma) by human lymphocytes in vivo and in vitro. METHODS: We used an AT(1)R blocker losartan in a randomized clinical trial in kidney transplant recipients over a 12-month period [AT(1)R blocker (N= 11) and control (N= 10)]. Peripheral blood lymphocytes, isolated from both cohorts, were analyzed by enzyme-linked immunosorbent spot assays (ELISPOT) analyses and real-time reverse transcription-polymerase chain reaction (RT-PCR) to enumerate IFN-gamma producing T cells and IFN-gamma mRNA levels. The effects of AT(1)R blockade in vitro were assessed using human alloreactive T cells and an IFN-gamma producing human cytotoxic T-lymphocyte line. Alloreactive T cells were treated with losartan or candesartan and enzyme-linked immunosorbant assay (ELISA) was used to measure IFN-gamma protein release. The cytotoxic T-lymphocyte line also was AT(1)R blocker-treated prior to determining IFN-gamma producing cells by intracellular cytokine staining. RESULTS: The AT(1)R blocker cohort had a significant decrease in IFN-gamma producing peripheral blood lymphocytes (P< or = 0.05 for each time point) and IFN-gamma mRNA levels (P= 0.01 vs. control patients). Losartan also decreased IFN-gamma production (P < 0.001) in purified alloreactive T cells in vitro as did candesartan. Moreover, Ang II amplified IFN-gamma generation (P < 0.05) in alloreactive T cells while AT(1)R blocker treatment inhibited Ang II's effect (P < 0.04). AT(1)R blocker treatment furthermore also inhibited IFN-gamma production in the cytotoxic T-lymphocyte line. CONCLUSION: AT(1)R blockers may have a clinically relevant immunomodulatory role by blocking IFN-gamma production in T cells.

Cyclooxygenase-2 expression is up-regulated in obstructed human ureter.

PURPOSE: Prostanoids produce significant effects on ureteral function and are synthesized by cyclooxygenase (COX) enzymes. COX is found in the 2 isoforms COX-1 (a constitutive form) and COX-2 (an inducible form). Due to the side effects associated with COX-1 inhibition there is great interest in selective COX-2 inhibition. We determined if COX-2 messenger (m)RNA and protein expression are regulated during ureteral obstruction. MATERIALS AND METHODS: mRNA analysis was performed using excess ureteral segments from 6 patients undergoing reconstructive procedures for chronic ureteral obstruction and 8 (normal ureter) undergoing donor nephrectomy after providing informed consent. All ureteral segments were snap frozen in liquid nitrogen and stored at -70C. RNA was isolated from the segments using phenol extraction and complementary DNA was synthesized by reverse transcription with murine leukemia virus reverse transcriptase (Promega Corp., Madison, Wisconsin). Semiquantitative reverse transcriptase-polymerase chain reaction was performed using specific COX-2 gene primers with ribosomal S26 primers serving as the housekeeping gene. The polymerase chain reaction product was quantified by agarose gel electrophoresis and phospho-imaging. The ratio of COX-2-to-S26 mRNA was compared. Additional segments were homogenized and total protein was extracted, separated via sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. These membranes were Western blotted for COX-2 and glyceraldehyde-3-phosphate dehydrogenase (housekeeping protein) with specific primary and secondary antibodies. RESULTS: The mean ratio of COX-2-to-S26 mRNA plus or minus standard error at 20, 22 and 24 cycles of amplification was 0.22 +/- 0.04 in the 8 normal ureters compared with 1.01 +/- 0.21 in the 6 obstructed ureters (unpaired Student's t test p = 0.004). Similarly the mean ratio of COX-2-to-glyceraldehyde-3-phosphate dehydrogenase protein on immunoblotting was 0.15 +/- 0.02 in the 8 normal ureters compared with 0.59 +/- 0.10 in the 6 obstructed ureters (p = 0.003). CONCLUSIONS: These data indicate that COX-2 mRNA and protein levels are up-regulated in chronically obstructed human ureters. Using selective COX-2 inhibitors may be useful for treating prostanoid induced effects associated with ureteral obstruction.

Donor genomics influence graft events: the effect of donor polymorphisms on acute rejection and chronic allograft nephropathy.

BACKGROUND: Organs procured from deceased donors emanate from individuals with diverse genetic backgrounds. Donor organs, therefore, may vary in their response to injury and immune stimuli in a genetically determined manner. We assessed polymorphisms from 244 renal allograft donors to better understand the impact of donor polymorphisms on selected transplant outcomes. METHODS: Donor genomic DNA restriction fragment length polymorphisms were assayed for evidence of common cytokine [interleukin (IL)-2, IL-6, IL-10, tumor necrosis factor (TNF)-alpha, TGF-beta, interferon (IFN)-gamma] and chemokine (CCR2, CCR5) polymorphisms. Associations between donor polymorphisms and graft events were determined using chi-square, linear regression, and Kaplan-Meier analyses. RESULTS: Several genotypic polymorphisms demonstrated a modest association with acute rejection, including the transforming growth factor (TGF)-beta T/C codon 10 (P= 0.027) and the CCR5 G/A 59029 (P= 0.039) genes by chi-square analysis. Notably, the presence of the T allele in the IFN-gamma gene (+874) demonstrated a highly significant association with biopsy-proven chronic allograft nephropathy (P < 0.008). This association remained highly significant in a multiple linear regression model that incorporated biopsy-proven acute rejection as a covariate. CONCLUSION: These data suggest that many of the donor polymorphisms studied in this analysis may influence a recipient's immune response to a renal allograft. However, their greatest impact may be demonstrated in long-term outcomes.