Protective vascular coagulation in response to bacterial infection of the kidney is regulated by bacterial lipid A and host CD147.

Bacterial infection of the kidney leads to a rapid cascade of host protective responses, many of which are still poorly understood. We have previously shown that following kidney infection with uropathogenic Escherichia coli (UPEC), vascular coagulation is quickly initiated in local perivascular capillaries that protects the host from progressing from a local infection to systemic sepsis. The signaling mechanisms behind this response have not however been described. In this study, we use a number of in vitro and in vivo techniques, including intravital microscopy, to identify two previously unrecognized components influencing this protective coagulation response. The acylation state of the Lipid A of UPEC lipopolysaccharide (LPS) is shown to alter the kinetics of local coagulation onset in vivo. We also identify epithelial CD147 as a potential host factor influencing infection-mediated coagulation. CD147 is expressed by renal proximal epithelial cells infected with UPEC, contingent to bacterial expression of the α-hemolysin toxin. The epithelial CD147 subsequently can activate tissue factor on endothelial cells, a primary step in the coagulation cascade. This study emphasizes the rapid, multifaceted response of the kidney tissue to bacterial infection and the interplay between host and pathogen during the early hours of renal infection.

Uropathogenic Escherichia coli P and Type 1 fimbriae act in synergy in a living host to facilitate renal colonization leading to nephron obstruction.

The progression of a natural bacterial infection is a dynamic process influenced by the physiological characteristics of the target organ. Recent developments in live animal imaging allow for the study of the dynamic microbe-host interplay in real-time as the infection progresses within an organ of a live host. Here we used multiphoton microscopy-based live animal imaging, combined with advanced surgical procedures, to investigate the role of uropathogenic Escherichia coli (UPEC) attachment organelles P and Type 1 fimbriae in renal bacterial infection. A GFP+ expressing variant of UPEC strain CFT073 and genetically well-defined isogenic mutants were microinfused into rat glomerulus or proximal tubules. Within 2 h bacteria colonized along the flat squamous epithelium of the Bowman's capsule despite being exposed to the primary filtrate. When facing the challenge of the filtrate flow in the proximal tubule, the P and Type 1 fimbriae appeared to act in synergy to promote colonization. P fimbriae enhanced early colonization of the tubular epithelium, while Type 1 fimbriae mediated colonization of the center of the tubule via a mechanism believed to involve inter-bacterial binding and biofilm formation. The heterogeneous bacterial community within the tubule subsequently affected renal filtration leading to total obstruction of the nephron within 8 h. Our results reveal the importance of physiological factors such as filtration in determining bacterial colonization patterns, and demonstrate that the spatial resolution of an infectious niche can be as small as the center, or periphery, of a tubule lumen. Furthermore, our data show how secondary physiological injuries such as obstruction contribute to the full pathophysiology of pyelonephritis.

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.

Glomerular sieving coefficient of serum albumin in the rat: a two-photon microscopy study.

Recent studies of the sieving of serum albumin in the rat kidney using a two-photon microscope suggested that the glomerular sieving coefficient (GSC) of albumin is 0.034, much higher than earlier micropuncture determinations. In the present study, we critically evaluated the use of the two-photon microscope to measure the GSC of albumin in the Munich-Wistar rat in vivo. The albumin GSC averaged 0.004 (SD 0.004), n = 34 glomeruli, when determined with a Zeiss two-photon microscope system and 0.002 (SD 0.002), n = 5, when determined with an Olympus two-photon microscope system. These values are close to the lower limit of detection of GSC, which we estimate to be approximately 0.001-0.003. We identified several factors that were likely responsible for the higher albumin GSCs reported earlier using two-photon microscopy. These include animal conditions (i.e., low glomerular filtration rate) and failure to recognize the role of out-of-focus fluorescence in contaminating the fluorescence signal from the urinary space of Bowman's capsule. We observed that hypothermia plus dehydration or a low blood pressure led to an increased albumin GSC. High levels of illumination (high laser outputs) resulted in a falsely elevated albumin GSC. Use of external, instead of internal, photodetectors resulted in an exaggerated albumin GSC because of greater collection of out-of-focus fluorescence. In conclusion, the albumin concentration in the glomerular filtrate of the normal rat, determined by two-photon microscopy, is exceedingly low (5-10 mg/dl).

Bacterial infection-mediated mucosal signalling induces local renal ischaemia as a defence against sepsis.

Ascending urinary tract infections can cause extensive damage to kidney structure and function. We have used a number of advanced techniques including multiphoton microscopy to investigate the crucial early phases of uropathogenic Escherichia coli induced pyelonephritis within a living animal. Our results reveal a previously undescribed innate vascular response to mucosal infection, allowing isolation and eradication of the pathogen. The extremely rapid host response to mucosal infection was highlighted by the triggering of a cascade of events within 3-4 h. Epithelial signalling produced an increase in cellular O(2) consumption and affected microvascular flow by clotting, causing localized ischaemia. Subsequent ischaemic damage affected pathophysiology with actin re-arrangement and epithelial sloughing leading to paracellular bacterial movement. A denuded tubular basement membrane is shown to hinder immediate dissemination of bacteria, giving the host time to isolate the infection by clotting. Suppression of clotting by heparin treatment caused fatal urosepsis. Clinically these findings may be relevant in antibiotics delivery in pyelonephritis patients and to the use of anticoagulants in sepsis.

Real-time studies of the progression of bacterial infections and immediate tissue responses in live animals.

By combining intravital multiphoton microscopy and bacterial genetics we have developed a technique enabling real-time imaging of bacterial proliferation and tissue responses in a live animal. Spatial and temporal control of the infection process was achieved by microinjecting GFP(+)-expressing uropathogenic Escherichia coli (UPEC) into tubules of exteriorized kidneys in live rats. GFP(+) was introduced in the clinical UPEC strain CFT073 as a single-copy chromosomal gene fusion. Within hours, bacterial colonization was accompanied by marked ischaemic effects, perivascular leakage, loss of tubular integrity and localized recruitment of immune cells. The pathophysiology was altered in response to an isogenic bacterial strain lacking the exotoxin haemolysin, revealing the subtle and temporal roles of bacterial virulence factors in vivo. Microdissection and RNA extraction of the injected nephron allowed molecular analysis of prokaryotic and eukaryotic gene expression. The techniques described here can be applied to study the integrated cell communication evoked by a variety of bacterial pathogens, assisting in the design of strategies to combat bacterial infections.

Fluorescent labeling of renal cells in vivo.

In vivo fluorescence imaging, using confocal or multiphoton microscopes, provides a powerful method to analyze kidney function in experimental animals. In this review, the preparation used for physiological studies in rats is described. A variety of fluorescent probes are available to study glomerular permeability, renal blood flow, peritubular capillary permeability, cell ion concentrations, tubule transport properties, and the functional status of renal cells. We have recently used micropuncture techniques and an adenovirus vector to accomplish gene transfer into kidney tubule and endothelial cells; this new methodology will allow the dynamic study of fluorescently-labeled proteins. Two examples of the use of two-photon fluorescence microscopy to study renal pathophysiology, namely polycystic kidney disease and renal ischemia, are presented. Software is available to quantify data collected from in vivo imaging experiments and to construct 3-dimensional images of renal structures. Two-photon or confocal microscopy offers many opportunities for a better understanding of kidney function in health and disease.

Micropuncture gene delivery and intravital two-photon visualization of protein expression in rat kidney.

Understanding molecular mechanisms of pathophysiology and disease processes requires the development of new methods for studying proteins in animal tissues and organs. Here, we describe a method for adenoviral-mediated gene transfer into tubule or endothelial cells of the rat kidney. The left kidney of an anesthetized rat was exposed and the lumens of superficial proximal tubules or vascular welling points were microinfused, usually for 20 min. The microinfusion solution contained adenovirus with a cDNA construct of either 1) Xenopus laevis actin depolymerizing factor/cofilin [XAC; wt-green fluorescent protein (GFP)], 2) actin-GFP, or 3) GFP. Sudan black-stained castor oil, injected into nearby tubules, allowed us to localize the microinfused structures for subsequent visualization. Two days later, the rat was anesthetized and the kidneys were fixed for tissue imaging or the left kidney was observed in vivo using two-photon microscopy. Expression of GFP and GFP-chimeric proteins was clearly seen in epithelial cells of the injected proximal tubules and the expressed proteins were localized similarly to their endogenously expressed counterparts. Only a minority of the cells in the virally exposed regions, however, expressed these proteins. Endothelial cells also expressed XAC-GFP after injection of the virus cDNA construct into vascular welling points. An advantage of the proximal tubule and vascular micropuncture approaches is that only minute amounts of virus are required to achieve protein expression in vivo. This micropuncture approach to gene transfer of the virus cDNA construct and intravital two-photon microscopy should be applicable to study of the behavior of any fluorescently tagged protein in the kidney and shows promise in studying renal physiology and pathophysiology.

Dietary potassium citrate does not harm the pcy mouse.

Formation of multiple cysts in the kidneys occurs in several inherited diseases and often leads to terminal kidney failure. Because there is no definitive therapy to halt or slow the progression of renal cystic disease in people, numerous studies have examined possible therapies in animal models. Autosomal-dominant polycystic kidney disease (ADPKD) in the Han:SPRD rat is ameliorated when alkalinizing citrate salts are provided in drinking solutions. By contrast, pcy mice with cystic disease fare worse with the same treatment. We tested the hypothesis that pcy mice ingesting citrate salts in the feed would not be adversely affected by this treatment. Male homozygous pcy mice were given regular feed or 6% potassium citrate-supplemented feed and ad libitum access to water starting at 3 weeks of age. The survival curves of the treated and untreated mice were not significantly different. We conclude that treatment with potassium citrate in the feed does not affect the progression of renal cystic disease in the pcy mouse. This model closely resembles human adolescent nephronophthisis (NPHP3). Based on these findings, citrate treatment cannot be recommended for NPHP3. The fact that it did no harm, however, removes a significant barrier to its consideration as a therapy for ADPKD.

Two-photon in vivo microscopy of sulfonefluorescein secretion in normal and cystic rat kidneys.

Sulfonefluorescein (SF) is a fluorescent organic anion secreted by kidney proximal tubules. The purposes of this study were 1) to quantify accumulation of SF in normal and cystic rat kidneys in vivo and 2) to test whether SF accumulation could be used as a marker for cysts derived from proximal tubules. Male Munich-Wistar rats, normal Han:SPRD rats, and heterozygous Han:SPRD rats with autosomal-dominant polycystic kidney disease were anesthetized with Inactin and solutions containing SF were administered by constant intravenous infusion. In Munich-Wistar rats, SF fluorescence in the urinary space of Bowman's capsule averaged 0.15 +/- 0.04 (n = 17) times that of glomerular capillary plasma, consistent with extensive plasma protein binding of SF. In normal Han:SPRD rats, steady-state cell cytoplasm SF fluorescence in proximal tubule and distal tubule cells averaged, respectively, 2.7 +/- 1.4 (n = 99 tubules) and 0.2 +/- 0.2 (n = 17) times that of peritubular capillary plasma. No punctate SF fluorescence was seen in proximal tubule cell cytoplasm. Probenecid reduced proximal tubule cell SF fluorescence to 0.64 +/- 0.40 (n = 64) times that of plasma. Ureteral obstruction decreased the proximal tubule cell-to-lumen SF fluorescence gradient, suggesting that tubule fluid flow normally sweeps away secreted SF. In cystic kidneys, cysts derived from proximal tubules could be identified by their uptake of SF, but cell uptake was patchy. We conclude that in vivo two-photon microscopy is a powerful tool for quantifying glomerular and tubular handling of SF, and SF can be used to identify proximal tubule-derived cysts.

Dietary citrate treatment of polycystic kidney disease in rats.

Progression of autosomal-dominant polycystic kidney disease (ADPKD) in the heterozygous male Han:SPRD rat is dramatically slowed by ingestion of potassium or sodium citrate. This study examined the efficacy of delayed therapy with sodium citrate, the effect of sodium citrate therapy on kidney cortex levels of transforming growth factor-beta (TGF-beta), and the response to calcium citrate ingestion. Rats were provided with citrate salts in their food, and renal clearance, blood pressure, blood chemistry, and survival determinations were made. Sodium citrate therapy was most effective when started at age 1 month, and delay of therapy until age 3 months produced no benefit. Kidney cortex TGF-beta levels were elevated in 3- and 8-month-old rats with ADPKD, but not in 6-week-old rats. Sodium citrate treatment, started at age 1 month, lowered TGF-beta levels to normal in 3-month-old rats, but this is probably not the primary mechanism of citrate's beneficial effect. Calcium citrate had only a modest effect in preserving glomerular filtration rate. Effective treatment of ADPKD in this rat model requires early administration of a readily absorbed alkalinizing citrate salt. Existing data on ADPKD patients on vegetarian diets or with kidney stones should be studied in light of these findings.

Atubular glomeruli in a rat model of polycystic kidney disease.

BACKGROUND: Autosomal-dominant polycystic kidney disease (ADPKD) is associated with a progressive decline in glomerular filtration rate (GFR) that often leads to end-stage renal disease. The basis for this decline in GFR is poorly understood. METHODS: Glomeruli in heterozygous Han:SPRD rats with ADPKD and their normal litter mates were studied by light microscopy, using serial sectioning techniques. The connections of the renal corpuscles to proximal tubules were classified as normal, atrophied, or absent (atubular glomerulus). Renal corpuscles also were examined by scanning electron microscopy. Single nephron glomerular blood flows were determined using microspheres. RESULTS: In the kidneys of six-month-old rats with ADPKD, 50% of the glomeruli were atubular and another 26% were associated with atrophied neck segments; these glomeruli were most often smaller in size than normal. About 16% of the glomeruli were hypertrophied and had normal connections to proximal tubules. Sclerotic changes in cystic kidney glomeruli were usually mild or moderate, and belied the failure of glomerular function. Glomerular blood flow in the cystic kidneys averaged half of normal and was markedly heterogeneous; the majority of small glomeruli displayed very low blood flows and a few showed relatively high blood flows. Fewer glomerular abnormalities were found in rats treated for five months with potassium citrate in their drinking water. CONCLUSIONS: The diminished GFR in the rat with ADPKD can be accounted for largely by the formation of atubular glomeruli. Compensatory glomerular hypertrophy also is present and may contribute to the progression of the renal disease.

Functional studies of the kidney of living animals using multicolor two-photon microscopy.

Optical microscopy, when applied to living animals, provides a powerful means of studying cell biology in the most physiologically relevant setting. The ability of two-photon microscopy to collect optical sections deep into biological tissues has opened up the field of intravital microscopy to high-resolution studies of the brain, lens, skin, and tumors. Here we present examples of the way in which two-photon microscopy can be applied to intravital studies of kidney physiology. Because the kidney is easily externalized without compromising its function, microscopy can be used to evaluate various aspects of renal function in vivo. These include cell vitality and apoptosis, fluid transport, receptor-mediated endocytosis, blood flow, and leukocyte trafficking. Efficient two-photon excitation of multiple fluorophores permits comparison of multiple probes and simultaneous characterization of multiple parameters and yields spectral information that is crucial to the interpretation of images containing uncharacterized autofluorescence. The studies described here demonstrate the way in which two-photon microscopy can provide a level of resolution previously unattainable in intravital microscopy, enabling kinetic analyses and physiological studies of the organs of living animals with subcellular resolution.