Hepatocyte necroptosis induced by ischemic acute kidney injury in rats.

While ischemic acute kidney injury (IAKI) is known often to cause hepatic injury, little is known about necroptosis involved in the hepatic injury. The purposes of this study were to identify necroptosis involvement and observe morphological changes of hepatocytes in hepatic injury induced by IAKI in rats. Based on successfully established IAKI rat models, enzyme-linked immunosorbent assay illustrated a significant higher level of tumor necrosis factor a in serums of IAKI animals. Tumor necrosis factor receptor a (TNFRa) and receptor interacting protein kinase 3 (RIPk3) showed significant higher expressions in immunoblot analyses and positive hepatocytes of RIPk3 immunohistochemical staining were also evident in livers of IAKI rats. In addition, light microscopy revealed necrotic lesions that contain hepatocytes ongoing necroptosis besides necrotic cells in IAKI livers. Electron microscopy revealed at least three types of necrotic hepatocytes, they were edema necrosis, vacuolization necrosis, and necroptosis. Hepatocytes undergoing necroptosis had both necrosis and apoptosis morphological characteristics, they were necrosis cytoplasm and apoptosis-like nucleus. Among cellular organelles of hepatocyte with necrosis, membranous structures, such as cell membrane, endoplasmic reticular system, and mitochondria were more vulnerable to the stress of IAKI and deformed nucleuses varied in shape and lytic or pyknotic chromatin appearances were noted under insults of IAKI. In conclusion, hepatocyte undergoing necroptosis, RIPk3-mediated necroptosis partly contributes to hepatic necrosis induced by IAKI. Both membranous structures and nucleuses of hepatocyte were vulnerable to ischemic acute kidney injury.

Ultrastructural pathology of rat lung injury induced by ischemic acute kidney injury.

Ischemic acute kidney injury (AKI) is a common complication during inpatient hospitalization, and often induces acute lung injury (ALI). A lot of studies have concentrated on the relevance between AKI and ALI, but the underlying mechanisms of AKI- associated ALI have remained unclear until now. One reason is that evidence of the ultrastructural pathology of AKI-associated ALI has been scarce and needed to be accumulated. The aims of present study are to observe ultrastructural changes, and to reveal leukocyte trafficking of ALI induced by ischemic AKI in rats. For this purpose light microscopy (LM) and electron microscopy (EM), as well as morphometric analysis, were employed in present study. LM observations revealed distinct regions of collapsed alveoli, hemorrhage in alveoli, and interstitial edema in AKI-induced ALI. EM examinations provided facts that alveolar epithelial cells, including type I and type II cells, were necrotic, and endothelia cells undergoing apoptosis as well as interstitial cells undergoing necroptosis were noted in AKI lungs. In addition, shrinkage and decreased or disappeared lamellar bodies were evident in alveolar type II cell of AKI rat lungs. Leukocyte numerical density on area (NA) in AKI lungs was significantly more than that in sham lungs. Based on the morphological criteria from EM examinations and morphometric analysis, a conclusion was that necrosis, including necroptosis, and apoptosis were involved in damaged lung induced by AKI. And inflammation also contributed to acute lung injury of rats with AKI.

Ultrastructural observations of programmed cell death during metanephric development in mouse.

Previous studies revealed apoptosis as an only programmed cell death (PCD) during renal morphogenesis before alternative type of PCD, necroptosis were introduced. Evidences of non-apoptotic PCD during renal development were scarce and needed to be accumulated. The purpose of this study is to investigate whether non-apoptotic PCD is involved in and observe ultrastructural features of apoptotic cells or non-apoptotic PCD during metanephros development. For this purpose, light and transmission electron microscopy were used. The most significant finding to come out of this study was that necroptosis was observed during developing metanephros by electron microscopy. The results also provided another fact that apoptosis and necroptosis constituted the PCD during embryonic development of kidney in mouse. Compared to necroptosis, apoptosis was more predominantly evident throughout whole development period and in every compartment of metanephros except for proximal tubule. However, necroptosis was only exhibited in developing nephrons also except for proximal tubule. In addition, outcomes of PCD were related to morphogenetic features of metanephric development. Efferocytosis for apoptotic cell or bodies took place in each type cell and whole period of developing metanephros. Besides efferocytosis blood flow and urine flux were available to remove the corpses of PCD, especially PCD from developing nephrons. Our findings suggested that both apoptosis and necroptosis play important roles during nephrogenesis and observed three ways to clear the PCD cell: efferocytosis, blood flow, and urine flux.

Glomerular permeability to macromolecules in the Necturus kidney.

Many aspects of the glomerular filtration of macromolecules remain controversial, including the location of the major filtration barrier, the effects of electrical charge, and the reason the filtration barrier does not clog. We examined these issues in anesthetized Necturus maculosus, using fluorescently labeled probes and a two-photon microscope. With the high resolution of this system and the extraordinary width ( approximately 3.5 mum) of the glomerular basement membrane (GBM) in this salamander, we were able to visualize fluorescent molecules in the GBM in vivo. GBM/plasma concentration ratios for myoglobin, ovalbumin, and serum albumin did not differ from that of inulin, indicating that the GBM does not discriminate among these molecules. The GBM/plasma concentration ratios for fluoresceinated dextran 500 and 2,000 kDa were significantly below that of inulin. Glomerular sieving coefficients (GSCs) for various macromolecules decreased as molecular mass increased, and the GSCs for bovine or human serum albumin were extremely low. The effect of electrical charge on filterability of a macromolecule was also examined. The GSCs for native (anionic) and neutral human serum albumin were not significantly different, nor did GSCs for anionic and neutral dextran 40 kDa differ, indicating that charge has no detectable effect on filterability of these macromolecules. These studies indicate that the main filtration barrier to albumin is the podocyte slit diaphragm. Electron microscopic studies revealed many cell processes within the GBM. Macromolecules that penetrated the GBM were taken up by mesangial cells and endothelial cells, suggesting that these cells help to prevent clogging of the filter.

Histopathology and surgical anatomy of patients with primary hyperparathyroidism and calcium phosphate stones.

Using a combination of intra-operative digital photography and micro-biopsy we measured renal cortical and papillary changes in five patients with primary hyperparathyroidism and abundant calcium phosphate kidney stones. Major tissue changes were variable papillary flattening and retraction, dilation of the ducts of Bellini, and plugging with apatite deposits of the inner medullary collecting ducts and ducts of Bellini. Some of the papillae in two of the patients contained plentiful large interstitial deposits of Randall's plaque and where the deposits were most plentiful we found overgrowth of the attached stones. Hence, this disease combines features previously described in brushite stone formers--dilation, plugging of ducts and papillary deformity--with the interstitial plaque and stone overgrowth characteristic of routine idiopathic calcium oxalate stone formers, suggesting that these two patterns can coexist in a single patient.

Mechanism of formation of human calcium oxalate renal stones on Randall's plaque.

Although calcium oxalate (CaOx) renal stones are known to grow attached to renal papillae, and specifically to regions of papillae that contain Randall's plaque (interstitial apatite deposits), the mechanisms of stone overgrowth on plaque are not known. To investigate the problem, we have obtained biopsy specimens from two stone patients that included an attached stone along with its tissue base and have studied the ultrastructural features of the attachment point using light and transmission electron microscopy, Fourier transform infrared spectroscopy (mu-FTIR), and immunohistochemical analysis. The epithelium is disrupted at the attachment site. The denuded plaque that borders on the urinary space attracts an envelope of ribbon-like laminates of crystal and organic matrix arising from urine ions and molecules. Into the matrix of this ribbon grow amorphous apatite crystals that merge with and give way to the usual small apatite crystals imbedded in stone matrix; eventually CaOx crystals admix with apatite and become the predominant solid phase. Over time, urine calcium and oxalate ions gradually overgrow on the large crystals forming the attached stone.

Apatite plaque particles in inner medulla of kidneys of calcium oxalate stone formers: osteopontin localization.

BACKGROUND: We have previously shown that interstitial plaque particles appear first in the basement membranes of thin loops of Henle and then in the interstitial space. However, it is not known if the plaque in the basement membrane of thin loops of Henle is of the same or different form than the interstitial plaque. Thus our purpose here is to detail the structure of the interstitial and membrane-bound plaque and explore the relationship of plaque apatite to osteopontin, a well-known crystal-associated urine protein. METHODS: Deep papillary biopsy tissue was studied from all 15 calcium oxalate stone formers and four nonforming subjects that we previously reported on [Evan et al, J Clin Ivest, 2003]. Routine light and transmission electron microscopy (TEM) as well as light microscopy and TEM immunohistochemical localization of osteopontin antibody were performed on all 19 subjects. RESULTS: In the basement membrane, plaque particles are individual and appear laminated with alternating light regions of crystal and electron-dense organic layers. In the interstitium, individual particles are not abundant but are instead aggregated to form regions of attached particles and in some regions what appears to be a fusion or syncytium in which crystal islands float in an organic sea. By light microscopy immunohistochemistry, osteopontin was localized to cells of the loops of Henle and collecting ducts as well as on sites of plaque. By immunoelectron microscopy, osteopontin immunogold label was found mainly on the surfaces of apatite crystal phase, at the junction of the crystal/organic layers. A similar immunogold labeling pattern was seen in the particles forming the syncytial islands of interstitial plaque. CONCLUSION: If indeed we accept the hypothesis that apatite plaque may be an anchored site on which calcium oxalate stones form and grow, the present work makes clear that it is unlikely that the surface of plaque presented to the final urine will be apatite crystal per se. However, our findings clearly show osteopontin is one of the crystal-associated urine proteins involved in the formation of the organic layers of the plaque particles.

Shockwave lithotripsy: dose-related effects on renal structure, hemodynamics, and tubular function.

BACKGROUND AND PURPOSE: Shockwave lithotripsy (SWL) predictably damages renal tissue and transiently reduces function in both kidneys. This study characterized the effects on renal function of a supraclinical dose of shockwaves (SWs) (8000) in porcine kidneys and tested the hypothesis that such excessive treatment would intensify and prolong the resulting renal impairment. MATERIALS AND METHODS: Pigs aged 6 to 7 weeks were anesthetized and assigned to one of three groups. Groups 1 (N=8) and 2 (N=6) each received 8000 SWs at 24 kV (Dornier HM3) to the lower-pole calix of one kidney. Group 3 (7 pigs) received sham treatment. Renal function was monitored for the first 4 hours after SW treatment in Group 1 and for 24 hours in Group 2. Plasma renin activity was measured in Groups 2 and 3. RESULTS: The renal lesions produced by 8000 SWs comprised 13.8%+/-1.4% of the renal mass. In the 4-hour protocol, this injury was associated with marked reduction of the glomerular filtration rate (GFR), renal plasma flow (RPF), and urinary sodium excretion in both kidneys, although fractional sodium excretion was reduced only in the shocked kidneys. In the 24-hour protocol, GFR and RPF remained below baseline in shocked kidneys at 24 hours. Evidence of progressive ischemic injury was noted in shocked tissue at 24 hours after SW treatment. CONCLUSIONS: These findings support the hypothesis that the severity of the renal injury caused by SWL is related to the number of SWs administered and demonstrate the connection in this relation between renal structure and function.

Crystal-associated nephropathy in patients with brushite nephrolithiasis.

BACKGROUND: We have biopsied the renal cortex and papillae of patients who form brushite renal stones asking if this unusual stone type is associated with specific tissue changes. We contrasted these with biopsies of 15 calcium oxalate stone formers, three stone formers with intestinal bypass, and four normal subjects. METHODS: We studied all ten brushite stone formers treated with percutaneous nephrolithotomy (PNL) during the past 3 years using digital video imaging of renal papillae, and obtained cortical and papillary biopsies. Biopsies were analyzed by light and electron microscopy, microinfrared spectroscopy, and electron diffraction. RESULTS: Apatite crystals plugged scattered terminal collecting ducts whose cells were injured or dead, and surrounding interstitium inflamed and fibrotic. White papillary deposits of interstitial apatite particles, so called Randall's plaque, were also present. Glomerular changes and cortical tubular atrophy and interstitial fibrosis were moderate to severe. CONCLUSION: Brushite stone formers combine the interstitial plaque of calcium oxalate stone formers with the collecting duct apatite plugs found in stone formers with intestinal bypass. Collecting duct injury and interstitial fibrosis are severe. Prominent cortical fibrosis, tubule atrophy, and glomerular pathology seem secondary to the collecting duct plugging. We believe crystallization obstructs and destroys terminal collecting duct segments thereby damaging nephrons, perhaps via intranephronal obstruction, and producing a hitherto unrecognized renal disease.

Morphological changes induced in the pig kidney by extracorporeal shock wave lithotripsy: nephron injury.

While shock wave lithotripsy (SWL) is known to cause significant damage to the kidney, little is known about the initial injury to cells along the nephron. In this study, one kidney in each of six juvenile pigs (6-7 weeks old) was treated with 1,000 shock waves (at 24 kV) directed at a lower pole calyx with an unmodified HM-3 lithotripter. Three pigs were utilized as sham-controls. Kidneys were fixed by vascular perfusion immediately after SWL or sham-SWL. Three of the treated kidneys were used to quantitate lesion size. Cortical and medullary samples for light (LM) and transmission electron microscopy (TEM) were taken from the focal zone for the shock waves (F2), the contralateral kidney, and the kidneys of sham-SWL pigs. Because preservation of the tissue occurred within minutes of SWL, the initial injury caused by the shock waves could be separated from secondary changes. No tissue damage was observed in contralateral sham-SWL kidneys, but treated kidneys showed signs of injury, with a lesion of 0.2% +/- 0.1% of renal volume. Intraparenchymal hemorrhage and injury to tubules was found at F2 in both the cortex and medulla of SWL-treated kidneys. Tubular injury was always associated with intraparenchymal bleeding, and the range of tissue injury included total destruction of tubules, focal cellular fragmentation, necrosis, cell vacuolization, and membrane blebbing. The initial injury caused by SWL was cellular fragmentation and necrosis. Cellular vacuolization, membrane blebbing, and disorganization of apical brush borders appear to be secondary changes related to hypoxia.

Randall's plaque of patients with nephrolithiasis begins in basement membranes of thin loops of Henle.

Our purpose here is to test the hypothesis that Randall's plaques, calcium phosphate deposits in kidneys of patients with calcium renal stones, arise in unique anatomical regions of the kidney, their formation conditioned by specific stone-forming pathophysiologies. To test this hypothesis, we performed intraoperative biopsies of plaques in kidneys of idiopathic-calcium-stone formers and patients with stones due to obesity-related bypass procedures and obtained papillary specimens from non-stone formers after nephrectomy. Plaque originates in the basement membranes of the thin loops of Henle and spreads from there through the interstitium to beneath the urothelium. Patients who have undergone bypass surgery do not produce such plaque but instead form intratubular hydroxyapatite crystals in collecting ducts. Non-stone formers also do not form plaque. Plaque is specific to certain kinds of stone-forming patients and is initiated specifically in thin-limb basement membranes by mechanisms that remain to be elucidated.

Kidney damage and renal functional changes are minimized by waveform control that suppresses cavitation in shock wave lithotripsy.

PURPOSE: In studies to understand better the role of cavitation in kidney trauma associated with shock wave lithotripsy we assessed structural and functional markers of kidney injury when animals were exposed to modified shock waves (pressure release reflector shock pulses) that suppress cavitation. Experiments were also performed in isolated red blood cells, an in vitro test system that is a sensitive indicator of cavitation mediated shock wave damage. MATERIALS AND METHODS: We treated 6-week-old anesthetized pigs with shock wave lithotripsy using an unmodified HM3 lithotriptor (Dornier Medical Systems, Marietta, Georgia) fitted with its standard brass ellipsoidal reflector (rigid reflector) or with a pressure release reflector insert. The pressure release reflector transposes the compressive and tensile phases of the lithotriptor shock pulse without otherwise altering the positive pressure or negative pressure components of the shock wave. Thus, with the pressure release reflector the amplitude of the incident shock wave is not changed but cavitation in the acoustic field is stifled. The lower pole of the right kidney was treated with 2,000 shocks at 24 kV. Glomerular filtration rate, renal plasma flow and tubular extraction of para-aminohippurate were measured in the 2 kidneys 1 hour before and 1 and 4 hours after shock wave lithotripsy, followed by the removal of each kidney for morphological analysis. In vitro studies assessed shock wave induced lysis to red blood cells in response to rigid or pressure release reflector shock pulses. RESULTS: Sham shock wave lithotripsy had no significant effect on kidney morphology, renal hemodynamics or para-aminohippurate extraction. Shock waves administered with the standard rigid reflector induced a characteristic morphological lesion and functional changes that included bilateral reduction in renal plasma flow, and unilateral reduction in the glomerular filtration rate and para-aminohippurate extraction. When the pressure release reflector was used, the morphological lesion was limited to hemorrhage of vasa recta vessels near the tips of renal papillae and the only change in kidney function was a decrease in the glomerular filtration rate at the 1 and 4-hour periods in shock wave treated kidneys. Red blood cell lysis in vitro was significantly lower with the pressure release reflector than with the rigid reflector. CONCLUSIONS: These data demonstrate that shock wave lithotripsy damage to the kidney is reduced when cavitation is suppressed. This finding supports the idea that cavitation has a prominent role in shock wave lithotripsy trauma.