Objective

Glomerular capillary hemorrhage (GCH) has been reported and confirmed as a consequence of contrast-enhanced diagnostic ultrasound (CEDUS) of rat kidney. This study assessed renal tissue injury in the larger porcine model.

Methods

The right kidneys of anesthetized pigs were imaged in 8 groups of 4 pigs. A Vingmed System Five (General Electric Co. Cincinnati OH) was used at 1.5 MHz in B-mode to intermittently scan the kidney at 4 s intervals. A Sequoia 512 (Acuson, Mountain View CA) was used in the 1.5 MHz Cadence CPS mode with intermittent agent-clearance bursts at 4 s intervals. Kidneys were scanned transabdominally, or after laparotomy through a saline standoff. The Sequoia 512 probe was placed in contact with the kidney for one group. Definity (Lantheus Medical Imaging, N. Billerica, MA) was infused at 4 μl/kg/min (diluted 33:1 in saline) for 4 min during scanning.

Results

Blood-filled urinary tubules were evident on the kidney surface for all groups, except for the group with the probe in contact with the kidney. GCH was found by histology in 31.7 % ± 9.8 % of glomeruli in the center of the scan plane for 1.7 MPa transabdominal scanning and 1.5 % ± 2.9 % of glomeruli in sham samples (P<0.05). In addition, hematuria was detected after scanning, and tubular obstruction occurred in some nephrons.

Conclusion

Renal tissue damage was induced by CEDUS in the porcine model. This result, together with previous studies in rats, support an hypothesis that GCH would occur in humans from similar CEDUS.

Diabetic nephropathy (DN) is among the most lethal complications that occur in type 1 and type 2 diabetics. Podocyte dysfunction is postulated to be a critical event associated with proteinuria and glomerulosclerosis in glomerular diseases including DN. However, molecular mechanisms of podocyte dysfunction in the development of DN are not well understood. Here we have shown that activity of mTOR complex 1 (mTORC1), a kinase that senses nutrient availability, was enhanced in the podocytes of diabetic animals. Further, podocyte-specific mTORC1 activation induced by ablation of an upstream negative regulator (PcKOTsc1) recapitulated many DN features, including podocyte loss, glomerular basement membrane thickening, mesangial expansion, and proteinuria in nondiabetic young and adult mice. Abnormal mTORC1 activation caused mislocalization of slit diaphragm proteins and induced an epithelial-mesenchymal transition–like phenotypic switch with enhanced ER stress in podocytes. Conversely, reduction of ER stress with a chemical chaperone significantly protected against both the podocyte phenotypic switch and podocyte loss in PcKOTsc1 mice. Finally, genetic reduction of podocyte-specific mTORC1 in diabetic animals suppressed the development of DN. These results indicate that mTORC1 activation in podocytes is a critical event in inducing DN and suggest that reduction of podocyte mTORC1 activity is a potential therapeutic strategy to prevent DN.

Glomerular capillary hemorrhage (GCH) in rat kidney provided a model for assessing in vivo gas body efficacy in diagnostic or therapeutic applications of ultrasound. Two diagnostic ultrasound machines were utilized: one monitored the harmonic B-mode contrast-enhancement of the left kidney and the other exposed the right kidney for GCH production. Definity contrast agent was infused at 1, 2, 5 or 10 μl/kg/min and infusion durations were 30, 60, 120 or 300 s. Exposure of the right kidney was at a peak rarefactional pressure amplitude of 2.3 MPa at 1.5 MHz. The circulating dose was estimated with a simple model of agent dilution and gas body loss. For 300 s infusion at 5 μl/kg/min, the left kidney image brightness increased to a plateau with an estimated 6.4±1.3 μl/kg circulating dose with no GCH in histological sections. Exposure of the right kidney with a 1 s image interval reduced the estimated circulating dose to 1.3±0.3 μl/kg and induced 68.4 % GCH. Dose and duration increases gave rapidly diminishing treatment effectiveness per gas body. The effective in vivo agent dose in rats can be reduced greatly due to high gas body destruction in the small animal, complicating predictions for similar conditions of human treatment.

Glomerular capillary hemorrhage (GCH) induced by ultrasonic cavitation during diagnostic imaging represents a unique contrast-agent related nephron injury. Consequences of GCH during 1.5 MHz diagnostic ultrasound with contrast agent were examined by histological methods in rats. Definity was infused at 10 μl/kg/min for 5 min at the start of 8 min of intermittent image-exposure with 2.3 MPa in situ peak rarefactional pressure amplitude. Kidney samples were taken for histology at 5 min, 30 min, 4 h, 2 d, 1 wk and 4 wks post exposure. In addition, samples were taken at 4 h from groups treated with heparin or aminocaproic acid. GCH was found in 61% of glomeruli in the center of the scan plane 5 min after exposure, which declined (P<0.05) to 36.3 % after 4 hr. The width of Bowman’s space was significantly increased for glomeruli with GCH relative to glomeruli without GCH (P<0.05), consistent with tubular obstruction. Antibody staining revealed fibrin clotting in Bowman’s space in 4 hr samples and this persisted in the 2 d samples. Heparin reduced and aminocaproic acid increased the GCH seen in 4 h samples. Tubular dilatation was evident with injury to the epithelium after 2d. After 1 week, areas of inflammatory cell infiltration were present. After 4 weeks, areas of interstitial fibrosis were revealed by Masson’s trichrome stain. The consequences of GCH induced by diagnostic ultrasound with contrast agents include rupture of glomerular capillaries, procoagulant activity resulting in intra-tubular obstruction, and the potential for progression of the resulting tubular injury toward interstitial fibrosis.

The autosomal recessive kidney disease nephronophthisis (NPHP) constitutes the most frequent genetic cause of terminal renal failure in the first 3 decades of life. Ten causative genes (NPHP1–NPHP9 and NPHP11), whose products localize to the primary cilia-centrosome complex, support the unifying concept that cystic kidney diseases are “ciliopathies”. Using genome-wide homozygosity mapping, we report here what we believe to be a new locus (NPHP-like 1 [NPHPL1]) for an NPHP-like nephropathy. In 2 families with an NPHP-like phenotype, we detected homozygous frameshift and splice-site mutations, respectively, in the X-prolyl aminopeptidase 3 (XPNPEP3) gene. In contrast to all known NPHP proteins, XPNPEP3 localizes to mitochondria of renal cells. However, in vivo analyses also revealed a likely cilia-related function; suppression of zebrafish xpnpep3 phenocopied the developmental phenotypes of ciliopathy morphants, and this effect was rescued by human XPNPEP3 that was devoid of a mitochondrial localization signal. Consistent with a role for XPNPEP3 in ciliary function, several ciliary cystogenic proteins were found to be XPNPEP3 substrates, for which resistance to N-terminal proline cleavage resulted in attenuated protein function in vivo in zebrafish. Our data highlight an emerging link between mitochondria and ciliary dysfunction, and suggest that further understanding the enzymatic activity and substrates of XPNPEP3 will illuminate novel cystogenic pathways.

This study was performed to examine the frequency dependence of glomerular capillary hemorrhage (GCH) induced by contrast aided diagnostic ultrasound in rats. Diagnostic ultrasound (DUS) scanners were utilized for exposure at 3.2, 5.0 and 7.4 MHz, and previously published data at 1.5 and 2.5 MHz also was included. A laboratory exposure system was used to simulate DUS exposure at 1.0, 1.5, 2.25, 3.5, 5.0 and 7.5 MHz with higher peak rarefactional pressure amplitudes (PRPAs) than were available from our DUS systems. The right kidneys of rats mounted in a water bath were exposed to intermittent image pulse sequences at 1 s intervals during infusion of diluted ultrasound contrast agent. The percentage of GCH was zero for low PRPAs, and then rapidly increased with increasing PRPAs above an apparent threshold, pt. The values of pt were approximately proportional to the ultrasound frequency, f, such that pt/f was approximately 0.5 MPa/MHz for DUS and 0.6 MPa/MHz for laboratory-system exposures. The increasing thresholds with increasing frequency limited the GCH effect for contrast aided DUS, and no GCH was seen for DUS at 5.0 or 7.4 MHz for the highest available PRPAs.

The right kidney of anaesthetized rats was imaged with intermittent diagnostic ultrasound (1.5 MHz; 1 sec trigger interval) under exposure conditions simulating those encountered in human perfusion imaging. The rats were infused intravenously with 10 μl/kg/min Definity® while being exposed to Mechanical Index (MI) values of up to 1.5 for 1 min. Suprathreshold MI values ruptured glomerular capillaries resulting in blood filling Bowman’s space and proximal convoluted tubules of many nephrons. The re-establishment of a pressure gradient following hemostasis caused the uninjured portions of the glomerular capillaries to resume the production of urinary filtrate which washed some or all of the erythrocytes out of Bowman’s space and cleared blood cells from some nephrons into urine within six hours. However, many of the injured nephrons remained plugged with tightly packed red cell casts 24 hours after imaging and also showed degeneration of tubular epithelium indicative of acute tubular necrosis. The additional damage caused by the extravasated blood amplified that caused by the original cavitating gas body. Human nephrons are virtually identical to those of the rat and so it is probable that similar glomerular capillary rupture followed by transient blockage and/or epithelial degeneration will occur following clinical exposures using similar high MI intermittent imaging with gas body contrast agents. The detection of blood in post-imaging urine samples using standard hematuria tests would confirm whether or not clinical protocols need to be developed to avoid this potential for iatrogenic injury.

Membrane-associated guanylate kinase (Maguk) proteins are scaffold proteins that contain PSD-95–Discs Large–zona occludens-1 (PDZ), Src homology 3, and guanylate kinase domains. A subset of Maguk proteins, such as mLin-2 and protein associated with Lin-7 (Pals)1, also contain two L27 domains: an L27C domain that binds mLin-7 and an L27N domain of unknown function. Here, we demonstrate that the L27N domain targets Pals1 to tight junctions by binding to a PDZ domain protein, Pals1-associated tight junction (PATJ) protein, via a unique Maguk recruitment domain. PATJ is a homologue of Drosophila Discs Lost, a protein that is crucial for epithelial polarity and that exists in a complex with the apical polarity determinant, Crumbs. PATJ and a human Crumbs homologue, CRB1, colocalize with Pals1 to tight junctions, and CRB1 interacts with PATJ albeit indirectly via binding the Pals1 PDZ domain. In agreement, we find that a Drosophila homologue of Pals1 participates in identical interactions with Drosophila Crumbs and Discs Lost. This Drosophila Pals1 homologue has been demonstrated recently to represent Stardust, a crucial polarity gene in Drosophila. Thus, our data identifies a new multiprotein complex that appears to be evolutionarily conserved and likely plays an important role in protein targeting and cell polarity.

Glomerular epithelial protein 1 (GLEPP1) is a receptor tyrosine phosphatase present on the apical cell surface of the glomerular podocyte. The GLEPP1 gene (Ptpro) was disrupted at an exon coding for the NH2-terminal region by gene targeting in embryonic stem cells. Heterozygote mating produced the expected genotypic ratio of 1:2:1, indicating that the Ptpro–/– genotype does not lead to embryonic or neonatal lethality. Kidney and glomerular structure was normal at the gross and light microscopic levels. Scanning and transmission electron microscopy showed that Ptpro–/– mice had an amoeboid rather than the typical octopoid structure seen in the wild-type mouse podocyte and that there were blunting and widening of the minor (foot) processes in association with altered distribution of the podocyte intermediate cytoskeletal protein vimentin. Reduced filtration surface area in association with these structural changes was confirmed by finding reduced glomerular nephrin content and reduced glomerular filtration rate in Ptpro–/– mice. There was no detectable increase in the urine albumin excretion of Ptpro–/– mice. After removal of one or more kidneys, Ptpro–/– mice had higher blood pressure than did their wild-type littermates. These data support the conclusion that the GLEPP1 (Ptpro) receptor plays a role in regulating the glomerular pressure/filtration rate relationship through an effect on podocyte structure and function.

Rabbit Hageman factor was proteolytically cleaved and activated by a homogenate prepared from cultured rabbit endothelial cells. Cleavage of radiolabeled Hageman factor was monitored by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Endothelial cell-mediated cleavage of Hageman factor was demonstrated both in a purified system and in plasma, was time and concentration dependent, and was associated with formation of the characteristic 28,000 Mr form of active Hageman factor. The rate of cleavage of Hageman factor was not affected by Triton X-100 (Rohm and Haas, Co., Philadelphia, Pa.), hexadimethrine bromide (Polybrene, Aldrich Chemical Co., Inc., Milwaukee, Wis.), hirudin, soybean trypsin inhibitor, or antisera to plasminogen or prekallikrein. However, cleavage was enhanced by kaolin, and was inhibited by diisopropyl-fluorophosphate. The enzyme responsible for cleavage of Hageman factor was localized to the 100,000-g-sedimentable, subcellular fraction of the endothelial cell homogenate and was relatively specific, because neither radiolabeled rabbit Factor XI nor rabbit prekallikrein were themselves proteolytically cleaved by the endothelial cell homogenate. However, when these molecules were incubated with the homogenate in the presence of Hageman factor, both Factor XI and prekallikrein were cleaved, demonstrating that Hageman factor had been activated by the endothelial cell homogenate. Furthermore, the kallikrein generated by endothelial cell homogenate-activated Hageman factor was capable of liberating kinin from high molecular weight kininogen as measured by bioassay. Cultured rabbit endothelial cells, therefore, possess the capacity to activate Hageman factor by proteolysis. This may be one mechanism for Hageman factor activation in vivo.

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