Peter K. Stricklett

Molecular Basis for Up-Regulation by Inflammatory Cytokines of Shiga Toxin 1 Cytotoxicity and Globotriaosylceramide Expression

Mortality in postdiarrheal hemolytic-uremic syndrome (HUS) is associated with brain injury. Normally, brain cells are resistant to Shiga toxin (Stx), the putative pathogenic toxin in HUS. However, exposure of human brain endothelial cells (HBECs) to tumor necrosis factor (TNF) and/or interleukin (IL)-1 markedly up-regulates Stx receptor (globotriaosylceramide; Gb3) expression and cytotoxicity. To investigate how Gb3 is augmented, ceramide glucosyltransferase (CGT), lactosylceramide synthase (GalT2), Gb3 synthase (GalT6), and alpha-galactosidase were studied in HBECs exposed to TNF and IL-1. TNF, both alone and in combination with IL-1, increased Stx-1 toxicity, Gb3 content, and Stx-1 binding. TNF in combination with IL-1 increased CGT, GalT2, and GalT6 but did not change alpha-galactosidase activities or mRNA levels. Cytokine treatment did not change CGT, GalT2, or GalT6 mRNA half-lives. Thus, inflammatory cytokine up-regulation of the sensitivity of HBECs to Stx-1 is the result of up-regulation, most likely via transcription, of the activities of 3 enzymes involved in Gb3 synthesis.

Molecular Basis for High Renal Cell Sensitivity to the Cytotoxic Effects of Shigatoxin-1: Upregulation of Globotriaosylceramide Expression

Cellular injury in post-diarrheal hemolytic-uremic syndrome (D+HUS) is related to shigatoxin (Stx) binding to globotriaosylceramide (Gb3). High renal Gb3 expression may determine renal susceptibility in D+HUS; however, the molecular mechanism(s) responsible for such relatively abundant Gb3 levels are unknown. Consequently, kidney cells expressing high Gb3 (cultured human proximal tubule cells [HPT]) were compared with non-kidney cells with low Gb3 content (cultured human brain microvascular endothelial cells [HBEC]). HPT were much more sensitive to the cytotoxic and protein synthesis inhibitory effects of Stx-1; this correlated with Gb3 content and (125)I-Stx-1 binding. HPT had greater Gb3 synthase (GalT6) and lower alpha-galactosidase activities than HBEC, whereas lactosylceramide synthase (GalT2) activity was higher in HBEC. Ceramide glucosyltransferase (CGT) activity was similar between the two cell types. The higher HPT GalT6 activity was associated with increased GalT6 mRNA steady-state levels, but no difference in GalT6 mRNA half-life. The lower HPT alpha-galactosidase activity was associated with reduced alpha-galactosidase mRNA steady-state levels but no difference in alpha-galactosidase mRNA half-life. Higher HBEC GalT2 activity was associated with increased steady-state GalT2 mRNA levels. These studies suggest that high renal Gb3 expression is due to enhanced GalT6 gene transcription and reduced alpha-galactosidase gene transcription and occur despite relatively low GalT2 activity.

Shiga Toxin-1 Regulation of Cytokine Production by Human Glomerular Epithelial Cells

Background/Aims: Inflammatory cytokines may enhance renal injury in post-diarrheal hemolytic uremic syndrome (Stx HUS) by enhancing the cytotoxic effect of Shiga toxins (Stx). The sources of inflammatory cytokines in Stx HUS are unclear. Since Stx-1 potently inhibits protein synthesis by glomerular epithelial cells (GEC) and increases cytokine release by renal epithelial cells, we examined Stx-1 regulation of cytokine production by human GEC. Methods: Stx-1 (and cycloheximide (CHX), another protein synthesis inhibitor) cytotoxicity, protein synthesis inhibition, and effect on interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor (TNF) release and mRNA levels were determined. Results: Stx-1 alone had a modest stimulatory effect on inflammatory cytokine production by GEC that occurred at toxin concentrations ranging from minimal to 50% inhibition of protein synthesis. CHX, at concentrations that produced similar inhibition of protein synthesis, increased IL-1, IL-6, and TNF protein release and mRNA accumulation, but in a different time- and dose-dependent pattern than Stx. Lipopolysaccharide (LPS) did not change IL-1, but stimulated IL-6 and TNF production. LPS and Stx-1 combined stimulated production of all three cytokines to a greater extent than either toxin alone. Conclusion: These data indicate that: (1) Stx-1 alone modestly stimulates GEC inflammatory cytokine production; (2) LPS and Stx-1 combined can potently enhance GEC cytokine release, and (3) this action of Stx-1 may relate in part to inhibition of protein synthesis but cannot be fully attributed to this effect.

The Cre/loxP system and gene targeting in the kidney

The Cre/loxP and Flp/FRT systems mediate site-specific DNA recombination and are being increasingly utilized to study gene function in vivo. These systems allow targeted gene disruption in a single cell type in vivo, thereby permitting study of the physiological and pathophysiological impact of a given gene product derived from a particular cell type. In the kidney, the Cre/loxP system has been employed to achieve gene deletion selectively within principal cells of the collecting duct. Disruption of target genes in the collecting duct, such as endothelin-1 or polycystic kidney disease-1 (PKD1), could lead to important insights into the biological roles of these gene products. With selection of the appropriate renal cell-specific promoters, these recombination systems could be used to target gene disruption to virtually any renal cell type. Although transgenic studies utilizing these recombination systems are promising, they are in their relative infancy and can be time consuming and expensive and yield unanticipated results. It is anticipated that continued experience with these systems will produce an important tool for analyzing gene function in renal health and disease.The Cre/loxP and Flp/FRT systems mediate site-specific DNA recombination and are being increasingly utilized to study gene function in vivo. These systems allow targeted gene disruption in a single cell type in vivo, thereby permitting study of the physiological and pathophysiological impact of a given gene product derived from a particular cell type. In the kidney, the Cre/loxP system has been employed to achieve gene deletion selectively within principal cells of the collecting duct. Disruption of target genes in the collecting duct, such as endothelin-1 or polycystic kidney disease-1 (PKD1), could lead to important insights into the biological roles of these gene products. With selection of the appropriate renal cell-specific promoters, these recombination systems could be used to target gene disruption to virtually any renal cell type. Although transgenic studies utilizing these recombination systems are promising, they are in their relative infancy and can be time consuming and expensive and yield unanticipated results. It is anticipated that continued experience with these systems will produce an important tool for analyzing gene function in renal health and disease.

Inactivation of Pkd1 in principal cells causes a more severe cystic kidney disease than in intercalated cells

Renal cysts in autosomal dominant polycystic kidney disease arise from cells throughout the nephron, but there is an uncertainty as to whether both the intercalated cells (ICs) and principal cells (PCs) within the collecting duct give rise to cysts. To determine this, we crossed mice containing loxP sites within introns 1 and 4 of the Pkd1 gene with transgenic mice expressing Cre recombinase under control of the aquaporin-2 promoter or the B1 subunit of the proton ATPase promoter, thereby generating PC- or IC-specific knockout of Pkd1, respectively. Mice, that had Pkd1 deleted in the PCs, developed progressive cystic kidney disease evident during the first postnatal week and had an average lifespan of 8.2 weeks. There was no change in the cellular cAMP content or membrane aquaporin-2 expression in their kidneys. Cysts were present in the cortex and outer medulla but were absent in the papilla. Mice in which PKd1 was knocked out in the ICs had a very mild cystic phenotype as late as 13 weeks of age, limited to 1-2 cysts and confined to the outer rim of the kidney cortex. These mice lived to at least 1.5 years of age without evidence of early mortality. Our findings suggest that PCs are more important than ICs for cyst formation in polycystic kidney disease.

Characterization of vasopressin-responsive collecting duct adenylyl cyclases in the mouse

Little is known about collecting duct adenylyl cyclase (AC) isoforms or regulation in the mouse. We performed RT-PCR for AC isoforms 1-9 in microdissected cortical (CCD) and outer medullary (OMCD) and acutely isolated inner medullary (IMCD) collecting duct. All collecting duct regions contained AC3, AC4, and AC6 mRNA, while CCD and OMCD, but not IMCD, also contained AC5 mRNA. Acutely isolated IMCD expressed AC3, AC4, and AC6 proteins by Western blot analysis. The mIMCD3 cell line expressed AC2, AC3, AC4, AC5, and AC6 mRNA; M-1 CCD cells expressed AC2, 3, 4, and 6, while mpkCCD cell lines contained AC3, AC4, and AC6 mRNA. AVP stimulated cAMP accumulation in acutely isolated mouse IMCD; this was reduced by chelation of extracellular calcium (EGTA) and almost completely abolished by blockade of calmodulin (W-7). Blockade of calmodulin kinase with KN-93 or endoplasmic reticulum calcium ATPase (thapsigargin) also reduced the AVP response. A similar inhibitory effect of W-7, KN-93, and thapsigargin was seen on forskolin-stimulated cAMP content in acutely isolated mouse IMCD. These three agents had the same pattern of blockade of AVP- or forskolin-stimulated AC activity in acutely isolated rat IMCD. AVP responsiveness in primary cultures of mouse IMCD was also reduced by W-7, KN-93, and thapsigargin. Small interfering RNA (siRNA) designed to knock down AC3 or AC6 in primary cultured mouse IMCD significantly reduced AVP-stimulated cAMP accumulation. Together, these data are consistent with a role of AC3 and AC6 in the activation of mouse collecting duct by AVP.

Inhibition of p38 Mitogen-Activated Protein Kinase Ameliorates Cytokine Up-Regulated Shigatoxin-1 Toxicity in Human Brain Microvascular Endothelial Cells

Brain injury in hemolytic-uremic syndrome (HUS) may be enhanced by inflammatory cytokine up-regulation of endothelial cell sensitivity to shigatoxin (Stx). The present study investigated whether inflammatory cytokine up-regulation of Stx toxicity could be ameliorated by inhibiting candidate signal transduction pathways. Exposure of human brain endothelial cells (HBECs) to tumor necrosis factor (TNF) greatly increased Stx-1 and Stx-2 cytotoxicity; this was reduced by inhibition of p38 mitogen-activated protein kinase (MAPK), but not c-Jun kinase. SB203580, a specific inhibitor of p38 MAPK, reduced TNF-stimulated Stx cytotoxicity in HBECs, TNF-stimulated (125)Stx-1 binding to intact HBECs, the cellular content of Gb3 (galactose alpha 1,4, galactose ss 1,4, glucose-ceramide) (the Stx receptor), and TNF-stimulated Gb3 synthase and glucosylceramide synthase activities but did not affect lactosylceramide synthase activities or mRNA content. Thus, inhibition of p38 MAPK substantially reduces inflammatory cytokine up-regulation of Stx-receptor synthesis and cell-surface expression, thereby decreasing Stx cytotoxicity. Inhibition of p38 MAPK may be of therapeutic benefit in HUS.

Effect of pioglitazone on survival and renal function in a mouse model of polycystic kidney disease.

Background/Aims: Cystic epithelia in polycystic kidney disease display features similar to malignant cells. Thiazolidinediones have been shown to have anti-neoplastic properties, therefore we tested the hypothesis that pioglitazone reduces cyst formation, improves renal function, and prolongs survival in a mouse model of polycystic kidney disease. Methods: PC-Pkd1-KO mice, which have homozygous mutations of the Pkd1 gene in principal cells, were used. On the day after giving birth, mothers were fed standard mouse chow with or without pioglitazone (30 mg/kg chow). After weaning, the assigned diet was continued. At 1 month of age, blood pressure was measured and animals were sacrificed to determine kidney weight, body weight, and serum urea. Kidneys were evaluated for proliferation using Ki-67, apoptosis using TUNEL analysis, and cyst number using MRI. Survival was observed. Results: Pioglitazone did not alter renal function, cell proliferation, apoptosis, or cyst formation in animals with polycystic kidney disease, however it did increase survival. Pioglitazone reduced blood pressure in PC-Pkd1-KO, but not in controls. Conclusion: These findings suggest that pioglitazone may have a unique antihypertensive effect in polycystic kidney disease, and that such an effect may promote improved survival.

Identification of Two Nuclear Factor of Activated T-cells (NFAT)-response Elements in the 5′-Upstream Regulatory Region of the ET-1 Promoter

Collecting duct-derived ET-1 regulates salt excretion and blood pressure. We have reported the presence of an inner medullary collecting duct (IMCD)-specific enhancer region in the 5′-upstream ET-1 promoter (Strait, K. A., Stricklett, P. K., Kohan, J. L., Miller, M. B., and Kohan, D. E. (2007) Am. J. Physiol. Renal Physiol. 293, F601–F606). The current studies provide further characterization of the ET-1 5′-upstream distal promoter to identify the IMCD-specific enhancer elements. Deletion studies identified two regions of the 5′-upstream ET-1 promoter, −1725 to −1319 bp and −1319 to −1026 bp, which were required for maximal promoter activity in transfected rat IMCD cells. Transcription factor binding site analysis of these regions identified two consensus nuclear factor of activated T-cells (NFAT) binding sites at −1263 and −1563. EMSA analysis using nuclear extracts from IMCD cells showed that both the −1263 and the −1563 NFAT sites in the ET-1 distal promoter competed for NFAT binding to previously identified NFAT sites in the IL-2 and TNF genes. Gel supershift analysis showed that each of the NFAT binding sites in the ET-1 promoter bound NFAT proteins derived from IMCD nuclear extracts, but they selectively bound different NFAT isoforms; ET-1263 bound NFATc1, whereas ET-1563 bound NFATc3. Site-directed mutagenesis of either the ET-1263 or the ET-1563 sites prevented NFAT binding and reduced ET-1 promoter activity. Thus, NFAT appears to be an important regulator of ET-1 transcription in IMCD cells, and thus, it may play a role in controlling blood pressure through ET-1 regulation of renal salt excretion.

Site-specific recombination using an epitope tagged bacteriophage P1 Cre recombinase

Since the original description of Cre mediated site-specific recombination in bacteriophage P1 (Sternberg, N., Hamilton, D., 1981 J. Mol. Biol., 150, 467-487), the Cre-lox system of recombination has been widely used to manipulate prokaryotic and eukaryotic genomes. Unfortunately, there are few means available to measure Cre protein expression in vivo. We have constructed an expression vector wherein the Cre protein is tagged at the carboxy terminus with an 11-amino-acid epitope to the herpes simplex virus (HSV) glycoprotein D coat protein (Isola, V.J., Eisenberg, R.J., Siebert, G.R., Heilman, C.J., Wilcox, W.C., Cohan, G.H., 1989. J. Virol. 63, 2325-2334). The epitope tag facilitates detection of Cre expression in vitro and in vivo using immunofluorescent labeling with a commercially available antibody. The epitope tag does not interfere with Cre recombinase activity or alter recombination efficiency between loxP sites. We have shown in mice that a transgene expressing our tagged Cre is capable of excising a loxP flanked sequence contributed by another transgenic mouse. In summary, we have developed an epitope-tagged Cre recombinase that is fully active and readily detectable.