George A. Tanner

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 Bowmans 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 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 Bowmans 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 O2 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.

Electrical Forces Determine Glomerular Permeability

There is ongoing controversy about the mechanisms that determine the characteristics of the glomerular filter. Here, we tested whether flow across the glomerular filter generates extracellular electrical potential differences, which could be an important determinant of glomerular filtration. In micropuncture experiments in Necturus maculosus, we measured a potential difference across the glomerular filtration barrier that was proportional to filtration pressure (-0.045 mV/10 cm H₂O). The filtration-dependent potential was generated without temporal delay and was negative within Bowmans space. Perfusion with the cationic polymer protamine abolished the potential difference. We propose a mathematical model that considers the relative contributions of diffusion, convection, and electrophoretic effects on the total flux of albumin across the filter. According to this model, potential differences of -0.02 to -0.05 mV can induce electrophoretic effects that significantly influence the glomerular sieving coefficient of albumin. This model of glomerular filtration has the potential to provide a mechanistic theory, based on experimental data, about the filtration characteristics of the glomerular filtration barrier. It provides a unique approach to the microanatomy of the glomerulus, renal autoregulation, and the pathogenesis of proteinuria.

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.

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.

Effects of Potassium Citrate/Citric Acid Intake in a Mouse Model of Polycystic Kidney Disease

The kidney function in a model of autosomal dominant polycystic kidney disease (PKD), the Han:SPRD rat, is dramatically improved by chronic ingestion of a solution of potassium citrate and citric acid (KCitr). This study investigated whether this treatment would also be beneficial in the pcy/pcy mouse, a model of autosomal recessive PKD. Starting at 1 month of age, male CD-1 pcy/pcy and normal CD-1 mice were provided with a solution of 55 mM K3 citrate/67 mM citric acid or tap water to drink. The pcy/pcy mice on the KCitr solution failed to grow normally and showed elevated plasma urea levels when compared to water-drinking littermates. Growth of normal CD-1 mice was not affected by KCitr intake. The pcy/pcy mice were then provided with a more dilute solution of KCitr to drink: this resulted in greater kidney wet and dry weights and a higher kidney weight/body weight ratio, but no beneficial effects. We conclude that pcy/pcy mice cannot tolerate a high level of KCitr intake and that a lower level is of no benefit. Whether KCitr therapy would be helpful in patients with PKD is still an open question.

Measurement of cell volume and fluid transport in MDCK cysts by video microscopy

A method for measuring cell volume and fluid transport rate in Madin-Darby canine kidney (MDCK) cysts is described. The method uses Nomarski optics and video microscopy to record changes in cyst dimensions in an in vitro superfusion system.

Reply: Podocytes are key—although albumin never reaches the slit diaphragm

We would like to thank Dr Comper for his correspondence on our Review (Renal albumin filtration: alternative models to the standard physical barriers. Nat. Rev. Nephrol. 9, 266–277; 2013)1 and the others in the Nature Reviews Nephrology collec‐ tion, which raises two important issues that must be addressed (Albuminuria is con‐ trolled primarily by proximal tubules. Nat. Rev. Nephrol. 28 January 2014; doi:10.1038/ nrneph.2013.58‐c1).2 F i r s t l y, D r C omp e r s t a t e s t h at “Nephrology students who read these articles would get the incorrect impres‐ sion that the glomerulus and/or podocytes are central to development of albumin‐ uria.” Contrary to what Comper proposes, the majority of researchers agree that the primary filtrate is virtually free of albumin; that is, the glomerular sieving coefficient (GSC) of albumin is extremely low (close to 0.001).3,4 Several lines of evidence support this position.1,3 For example, micropuncture studies in the rat kidney have demonstrated very low albumin concentrations in the primary filtrate. In cooled or fixed isolated perfused kidney, cubilin/megalin knockout mice or human patients with Fanconi syn‐ drome, only very little protein is excreted in the urine (<1 g albumin per day in humans). Additionally, filtered protein has been observed in Bowman’s space in histologi‐ cal sections only in proteinuric rodents, but not in normal controls. Other mam malian filtration systems also show a very low albumin sieving coefficient—for example, the choroid plexus in humans has a sieving coefficient of 0.003–0.0008. Finally, muta‐ tion of a podocyte‐specific gene (NPHS2, which encodes podocin) is sufficient to result in nephrotic‐range proteinuria. Why is the podocyte essential for glo‐ merular integrity? The glomerular filtration barrier challenges us with some formid‐ able ‘riddles’ and controversy abounds as to how it functions. One of these riddles is REPLY