The effect of mycophenolate mofetil on podocytes in nephrotoxic serum nephritis.

Mycophenolate mofetil (MMF) is applied in proteinuric kidney diseases, but the exact mechanism of its effect on podocytes is still unknown. Our previous in vitro experiments suggested that MMF can ameliorate podocyte damage via restoration of the Ca2+-actin cytoskeleton axis. The goal of this study was to characterize podocyte biology during MMF treatment in nephrotoxic serum (NTS) nephritis (NTN). NTN was induced in three-week old wild-type mice. On day 3, half of the mice were treated with MMF (100 mg/kgBW/d p.o.) for one week. On day 10, we performed proteomic analysis of glomeruli as well as super-resolution imaging of the slit diaphragm. For multiphoton imaging of Ca2+ concentration ([Ca2+]i), the experimental design was repeated in mice expressing podocyte-specific Ca2+ sensor. MMF ameliorated the proteinuria and crescent formation induced by NTS. We identified significant changes in the abundance of proteins involved in Ca2+ signaling and actin cytoskeleton regulation, which was further confirmed by direct [Ca2+]i imaging in podocytes showing decreased Ca2+ levels after MMF treatment. This was associated with a tendency to restoration of podocyte foot process structure. Here, we provide evidence that MPA has a substantial direct effect on podocytes. MMF contributes to improvement of [Ca2+]i and amelioration of the disorganized actin cytoskeleton in podocytes. These data extend the knowledge of direct effects of immunosuppressants on podocytes that may contribute to a more effective treatment of proteinuric glomerulopathies with the least possible side effects.

Protocol for a randomized controlled multicenter trial assessing the efficacy of leuprorelin for severe polycystic liver disease: the AGAINST-PLD study.

BACKGROUND: In patients with severe polycystic liver disease (PLD), there is a need for new treatments. Estrogens and possibly other female sex hormones stimulate growth in PLD. In some patients, liver volume decreases after menopause. Female sex hormones could therefore be a target for therapy. The AGAINST-PLD study will examine the efficacy of the GnRH agonist leuprorelin, which blocks the production of estrogen and other sex hormones, to reduce liver growth in PLD. METHODS: The AGAINST-PLD study is an investigator-driven, multicenter, randomized controlled trial. Institutional review board (IRB) approval was received at the University Medical Center of Groningen and will be collected in other sites before opening these sites. Thirty-six female, pre-menopausal patients, with a very large liver volume for age (upper 10% of the PLD population) and ongoing liver growth despite current treatment options will be randomized to direct start of leuprorelin or to 18 months standard of care and delayed start of leuprorelin. Leuprorelin is given as 3.75 mg subcutaneously (s.c.) monthly for the first 3 months followed by 3-monthly depots of 11.25 mg s.c. The trial duration is 36 months. MRI scans to measure liver volume will be performed at screening, 6 months, 18 months, 24 months and 36 months. In addition, blood will be drawn, DEXA-scans will be performed and questionnaires will be collected. This design enables comparison between patients on study treatment and standard of care (first 18 months) and within patients before and during treatment (whole trial). Main outcome is annualized liver growth rate compared between standard of care and study treatment. Secondary outcomes are PLD disease severity, change in liver growth within individuals and (serious) adverse events. The study is designed as a prospective open-label study with blinded endpoint assessment (PROBE). DISCUSSION: In this trial, we combined the expertise of hepatologist, nephrologists and gynecologists to study the effect of leuprorelin on liver growth in PLD. In this way, we hope to stop liver growth, reduce symptoms and reduce the need for liver transplantation in severe PLD. Trial registration Eudra CT number 2020-005949-16, registered at 15 Dec 2020. https://www.clinicaltrialsregister.eu/ctr-search/search?query=2020-005949-16 .

A KRAS variant is a biomarker of poor outcome, platinum chemotherapy resistance and a potential target for therapy in ovarian cancer.

Germline variants in the 3' untranslated region (3'UTR) of cancer genes disrupting microRNA (miRNA) regulation have recently been associated with cancer risk. A variant in the 3'UTR of the KRAS oncogene, referred to as the KRAS variant, is associated with both cancer risk and altered tumor biology. Here, we test the hypothesis that the KRAS variant can act as a biomarker of outcome in epithelial ovarian cancer (EOC), and investigate the cause of altered outcome in KRAS variant-positive EOC patients. As this variant seems to be associated with tumor biology, we additionally test the hypothesis that this variant can be directly targeted to impact cell survival. EOC patients with complete clinical data were genotyped for the KRAS variant and analyzed for outcome (n=536), response to neoadjuvant chemotherapy (n=125) and platinum resistance (n=306). Outcome was separately analyzed for women with known BRCA mutations (n=79). Gene expression was analyzed on a subset of tumors with available tissue. Cell lines were used to confirm altered sensitivity to chemotherapy associated with the KRAS variant. Finally, the KRAS variant was directly targeted through small-interfering RNA/miRNA oligonucleotides in cell lines and survival was measured. Postmenopausal EOC patients with the KRAS variant were significantly more likely to die of ovarian cancer by multivariate analysis (hazard ratio=1.67, 95% confidence interval: 1.09-2.57, P=0.019, n=279). Perhaps explaining this finding, EOC patients with the KRAS variant were significantly more likely to be platinum resistant (odds ratio=3.18, confidence interval: 1.31-7.72, P=0.0106, n=291). In addition, direct targeting of the KRAS variant led to a significant reduction in EOC cell growth and survival in vitro. These findings confirm the importance of the KRAS variant in EOC, and indicate that the KRAS variant is a biomarker of poor outcome in EOC likely due to platinum resistance. In addition, this study supports the hypothesis that these tumors have continued dependence on such 3'UTR lesions, and that direct targeting may be a viable future treatment approach.

[Treating the symptoms in nephrotic syndrome: which therapeutic strategies are evidence based in the treatment of proteinuria?]. / Symptomatische Therapie bei nephrotischem Syndrom: Was ist gesichert in der Therapie der Proteinurie?.

Glomerular diseases are among the most common renal pathologies leading frequently to end-stage renal disease. Clinical disease can be divided into five different groups the features of which are determined by the underlying pathophysiology. One of these five clinical syndromes is the nephrotic syndrome, which is characterized by proteinuria > 3.5 g/day accompanied by hypalbuminemia, hyperlipoproteinemia and pronounced edema. The nephrotic syndrome may be the clinical manifestation of a row of underlying diseases. The pathophysiological basics had remained elusive for decades, yet recently significant progress which allows for establishing new therapeutic strategies has been made. A major breakthrough in understanding the function of the glomerular filter unit has been possible in the last years through both genetic and cell biological studies, which have revealed a crucial role for the visceral epithelial cells of the glomerulus - the podocytes. By now various factors have been found causing podocyte damage, such as toxines, immunological phenomena or systemic disease like diabetes mellitus.

The mir-34 microRNA is required for the DNA damage response in vivo in C. elegans and in vitro in human breast cancer cells.

MicroRNAs (miRNAs) are important regulators of cell fate determination and homeostasis. Expression of these small RNA genes is tightly regulated during development and in normal tissues, but they are often misregulated in cancer. MiRNA expression is also affected by DNA damaging agents, such as radiation. In particular, mammalian miR-34 is upregulated by p53 in response to radiation, but little is known about the role of this miRNA in vivo. Here we show that Caenorhabditis elegans with loss-of-function mutations in the mir-34 gene have an abnormal cellular survival response to radiation; these animals are highly radiosensitive in the soma and radioresistant in the germline. These findings show a role for mir-34 in both apoptotic and non-apoptotic cell death in vivo, much like that of cep-1, the C. elegans p53 homolog. These results have been additionally validated in vitro in breast cancer cells, wherein exogenous addition of miR-34 alters cell survival post-radiation. These observations confirm that mir-34 is required for a normal cellular response to DNA damage in vivo resulting in altered cellular survival post-irradiation, and point to a potential therapeutic use for anti-miR-34 as a radiosensitizing agent in p53-mutant breast cancer.

Study of CA1 place cell activity and exploratory behavior following spatial and nonspatial changes in the environment.

Changes in the spatial arrangement or identity of objects inside a familiar environment induce reexploration. The present study looks at modifications of place cell activity during such renewed exploration. Hungry rats foraged for food in a cylinder with a salient cue card attached to the wall and with two distinct objects at fixed positions on the floor relative to each other and to the cue card. Once a set of CA1 place cells was recorded in this standard configuration, additional sessions were done after two kinds of manipulation. In the first, the two objects were rotated as a rigid set 90 degrees counterclockwise around the cylinder center while leaving the cue card in place; this was considered a spatial change. The effects of rotating the objects were different for fields near the objects (near fields) and fields far from the objects (far fields). Object rotation altered most near fields in complex ways, including remapping and cessation of firing. Near fields that remained intact after object rotation underwent unpredictable rotations that frequently departed considerably from the expected value of 90 degrees CCW. In contrast, the only change induced in far fields was a reduction of discharge rate on day 1, but not day 2, exposures of the rat to the rotated objects. The effects on both near and far fields were reversed when the objects were returned to their standard position. In the second manipulation, substitution of one of the two familiar objects with a novel object, a nonspatial change, had no detectable effect on place cell activity, regardless of field location. The sensitivity of hippocampal place cells to spatial changes but not to nonspatial changes is in agreement with earlier results showing that hippocampal lesions abolish reexploration after spatial but not after nonspatial object manipulations. The fact that reexploration is accompanied by place cell changes after spatial but not nonspatial changes reinforces the role that the hippocampus is believed to play in navigational computing and is perfectly compatible with the idea that another brain structure, likely perirhinal cortex, is responsible for object recognition.

Spatial navigation and hippocampal place cell firing: the problem of goal encoding.

Place cells are hippocampal neurons whose discharge is strongly related to a rat's location in the environment. The existence of such cells, combined with the reliable impairments seen in spatial tasks after hippocampal damage, has led to the proposal that place cells form part of an integrated neural system dedicated to spatial navigation. This hypothesis is supported by the strong relationships between place cell activity and spatial problem solving, which indicate that the place cell representation must be both functional and in register with the surroundings for the animal to perform correctly in spatial tasks. The place cell system nevertheless requires other essential elements to be competent, such as a component that specifies the overall goal of the animal and computes the path required to take the rat from its current location to the goal. Here, we propose a model of the neural network responsible for spatial navigation that includes goal coding and path selection. In this model, the hippocampal formation allows for place recognition, and stores the set of places that can be accessed from each position in the environment. The prefrontal cortex is responsible for encoding goal location and for route planning. The nucleus accumbens translates paths in neural space into appropriate locomotor activity that moves the animal towards the goal in real space. The complete model assumes that the hippocampal output to nucleus accumbens and prefrontal cortex provides information for generating solutions to spatial problems. In support of this model, we finally present preliminary evidence that the goal representation necessary for path planning might be encoded in the prelimbic/infralimbic region of the medial prefrontal cortex.

Quantifying location-specific information in the discharge of rat hippocampal place cells.

The informational content in the location-specific discharge of rat hippocampal cells is usually quantified by an average for the entire behaviorally accessible space. In contrast to such "global" information measures, we consider here information that can be obtained from "local" spike counts at each position. The properties of these local information measures are first illustrated using simulated data with predetermined distributions of location-specific spike counts. Next, place cell recordings from rats foraging in a cylindrical arena with two cue cards on its walls are analyzed; time windows as short as 100 ms were used to accumulate spike counts in locations. We show that information at the centers of firing fields is higher for fields nearer to the cues. Neither firing rates or "global" information measures detected differences between fields near and far from the cues. Thus, analyses of the location-specific information provides a new valuable tool for studying the location-specific activity of rat hippocampal cells. Generalizations of location-specific information can be used to investigate place cell responses to other factors such as running speed or the state of the hippocampal EEG in addition to current position.

Muscarinic blockade slows and degrades the location-specific firing of hippocampal pyramidal cells.

The firing of rat hippocampal pyramidal cells is determined both by the animal's location and by the state of the hippocampal EEG. Because cholinergic transmission plays a role in EEG activity, we expected that its modification would alter place cell activity. We therefore investigated the effects on place cell activity of blocking muscarinic transmission with intracerebroventricular injections of scopolamine. Scopolamine reduced both the rate of place cell discharge inside firing fields and the spatial coherence of the fields; discharge outside of the fields also showed small increases. After injections, fields were shifted farther from their previous location than for saline controls, indicating reduced reproducibility after muscarinic blockade. Scopolamine increased the time rats were stationary, but changes in place cell activity persisted even after analysis was restricted to periods of walking, suggesting that the behavioral changes cannot account for the cell discharge changes. The scopolamine effects were dose dependent to an extent that varied between different measures. The firing rates of interneurons showed only a minor trend to decrease after scopolamine. Nevertheless, the spatial coherence of interneuron firing patterns was reduced, consistent with the recent demonstration that their positional firing is mediated by the location-specific firing of pyramids (Marshall et al., 2002). These results demonstrate that acetylcholine enhances positional firing patterns in the hippocampus. Muscarinic blockade weakens the positional firing of most place cells and therefore renders them less useful for precise representation of the environment. This effect may underlie the difficulties in spatial learning and problem solving caused by abnormalities of cholinergic transmission.

Consolidation of extinction learning involves transfer from NMDA-independent to NMDA-dependent memory.

Extinction of conditioned fear to a tone paired with foot shock is thought to involve the formation of new memory. In support of this, previous studies have shown that extinction of conditioned fear depends on NMDA receptor-mediated plasticity. To further investigate the role of NMDA receptors in extinction, we examined the effects of the NMDA antagonist d(-)-3-(2-carboxypiperazine-4-yl)-propyl-1-phosphonic acid (CPP) on the extinction of conditioned freezing and suppression of bar pressing (conditioned emotional response). Rats extinguished normally during a 90 min session in the presence of systemic CPP (10 mg/kg), but were unable to recall extinction learning 24 hr later. This suggests that an NMDA-independent form of plasticity supports short-term extinction memory, but NMDA receptors are required for consolidation processes leading to long-term extinction memory. Surprisingly, extinction learned in the presence of CPP was recalled normally when tested 48 hr after training, suggesting a delayed consolidation process that was able to improve memory in the absence of further training. Delayed consolidation involves NMDA receptors because CPP injected on the rest day between training and test prevented 48 hr recall of extinction learned under CPP. Control experiments showed that the effect of CPP on memory consolidation was not caused by state-dependent learning or reduced expression of freezing under CPP. These findings demonstrate that NMDA receptor activation is critical for consolidation of extinction learning and that this process can be initiated after training has taken place. We suggest that consolidation of extinction involves off-line relearning that reinforces extinction memory through NMDA-mediated plasticity, perhaps in prefrontal-amygdala circuits.

Parallel instabilities of long-term potentiation, place cells, and learning caused by decreased protein kinase A activity.

To further elucidate the links among synaptic plasticity, hippocampal place cells, and spatial memory, place cells were recorded from wild-type mice and transgenic "R(AB)" mice with reduced forebrain protein kinase A (PKA) activity after introduction into a novel environment. Place cells in both strains were similar during the first exposure and were equally stable for recording sessions separated by 1 hr. Place cell stability in wild-type mice was unchanged for sessions separated by 24 hr but was reduced in R(AB) mice over the longer interval. This stability pattern parallels both the reduced late-phase long-term potentiation in hippocampal slices from R(AB) mice and the amnesia for context fear conditioning seen in R(AB) mice 24 but not 1 hr after training. The similar time courses of synaptic, network, and behavioral instability suggest that the genetic reduction of PKA activity is responsible for the defects at each level and support the idea that hippocampal synaptic plasticity is important in spatial memory.

Conjoint control of hippocampal place cell firing by two visual stimuli. I. The effects of moving the stimuli on firing field positions.

To better understand how hippocampal place cell activity is controlled by sensory stimuli, and to further elucidate the nature of the environmental representation provided by place cells, we have made recordings in the presence of two distinct visual stimuli under standard conditions and after several manipulations of these stimuli. In line with a great deal of earlier work, we find that place cell activity is constant when repeated recordings are made in the standard conditions in which the centers of the two stimuli, a black card and a white card, are separated by 135 degrees on the wall of a cylindrical recording chamber. Rotating the two stimuli by 45 degrees causes equal rotations of place cell firing fields. Removing either card and rotating the other card also causes fields to rotate equally, showing that the two stimuli are individually salient. Increasing or decreasing the card separation (card reconfiguration) causes a topological distortion of the representation of the cylinder floor such that field centers move relative to each other. We also found that either kind of reconfiguration induces a position-independent decrease in the intensity of place cell firing. We argue that these results are not compatible with either of two previously stated views of the place cell representation; namely, a nonspatial theory in which each place cell is tuned to an arbitrarily selected subset of available stimuli or a rigid map theory. We propose that our results imply that the representation is map-like but not rigid; it is capable of undergoing stretches without altering the local arrangement of firing fields.

Conjoint control of hippocampal place cell firing by two visual stimuli. Ii. A vector-field theory that predicts modifications of the representation of the environment.

Changing the angular separation between two visual stimuli attached to the wall of a recording cylinder causes the firing fields of place cells to move relative to each other, as though the representation of the floor undergoes a topological distortion. The displacement of the firing field center of each cell is a vector whose length is equal to the linear displacement and whose angle indicates the direction that the field center moves in the environment. Based on the observation that neighboring fields move in similar ways, whereas widely separated fields tend to move relative to each other, we develop an empirical vector-field model that accounts for the stated effects of changing the card separation. We then go on to show that the same vector-field equation predicts additional aspects of the experimental results. In one example, we demonstrate that place cell firing fields undergo distortions of shape after the card separation is changed, as though different parts of the same field are affected by the stimulus constellation in the same fashion as fields at different locations. We conclude that the vector-field formalism reflects the organization of the place-cell representation of the environment for the current case, and through suitable modification may be very useful for describing motions of firing patterns induced by a wide variety of stimulus manipulations.

Is the hippocampus of the rat part of a specialized navigational system?

The spatial mapping theory of hippocampal function proposes that the rat hippocampus is specialized for navigational computations, computations that allow the animal to solve difficult spatial problems. In this paper, we review evidence obtained by recording place cells and other "spatially tuned" cells from freely moving rats. Our main conclusion is that the nature of the signals carried by these cells and the ways in which the signals transform after changing the environment imply that the hippocampus and associated structures are able to represent aspects of the geometry of the environment.

Further study of the control of place cell firing by intra-apparatus objects.

The angular positions of hippocampal place cell firing fields are accurately controlled by the position of a single salient cue card attached to the wall of a recording cylinder; when the card is rotated, fields rotate equally. In contrast, the control exerted by 3-dimensional objects placed directly in the recording arena depends on their arrangement. When three objects lie on the vertices of an isosceles triangle near the center of the cylinder they rarely exert any control over the angular positions of firing fields. However, if the isosceles triangle is dilated so that its vertices are against the apparatus wall, the objects exert virtually ideal control over angular field position. Why do the objects gain control when they are against the cylinder wall? One possibility is that the asymmetry in the object set is more easily detected when the objects are far apart so that they provide a better polarizing cue. This hypothesis assumes that the identity of individual landmarks is not recognized by the place cell system whereas their geometric arrangement provides crucial information for controlling place field positions. If this is true, putting the 3 objects against the cylinder wall on the vertices of an equilateral triangle should cause a loss of stimulus control over the angular positions of firing fields. To the contrary, we found that the firing fields of most place cells (23/29) were accurately controlled by the equilateral object arrangement. Moreover, 5/6 of the uncontrolled cells were in a single animal. These results bolster our previous suggestion that the centrally placed objects fail to control place field positions because the computations necessary to form a stable reference frame are very difficult when the animal can go between stimuli.

Comparisons of head direction cell activity in the postsubiculum and anterior thalamus of freely moving rats.

Single cells in the rat anterior thalamic nucleus (ATN) and postsubiculum (PoS) discharge as a function of the rat's directional heading in the horizontal plane, independent of its location. A previous study that compared cell firing during clockwise and counterclockwise head turns concluded that ATN 'head direction' (HD) cell discharge anticipates the rat's future directional heading, while PoS HD cell discharge is in register with the rat's current directional heading (Blair and Sharp [1995] J Neurosci 15:6260-6270). In the current study we extend these findings by using a different method of analysis. HD cells in the ATN and PoS were first characterized by three different measures: peak firing rate, range width, and information content. We then examined how these measures varied when cell firing was aligned with past (negative time shift) or future (positive time shift) head direction of the rat. We report that all three measures were optimized when ATN cell firing was aligned with the animal's future directional heading by about +23 msec. In contrast, PoS HD cell firing was optimized when cell firing was aligned with the rat's past head direction by about -7 msec. When the optimal value was plotted as a function of the amount of time spikes were shifted relative to head orientation, the mean ATN function was shifted to the right of the PoS function only at negative time shifts; at positive time shifts the two functions overlapped. Analysis of two recording sessions from the same cell indicated that each cell in a particular brain area is 'tuned' to a specific time shift so that all cells within a brain area are not uniformly tuned to the same time shift. Other analyses showed that the clockwise and counterclockwise tuning functions were not skewed in the direction of the head turn as postulated by Redish et al. ([1996] Network: Computation in Neural Systems 7:671-685) and Blair et al. ([1997] J Neurophysiol 17:145-159). Additional analysis on episodes when the rat happened to continually point its head in the preferred direction indicated that HD cell firing undergoes little adaptation. In the Discussion, we argue that these results are best accounted for by a motor efference copy signal operating on both types of HD cells such that the copy associated with the PoS HD cells is delayed in time by about 30 msec relative to the copy associated with ATN HD cells.

Place cell discharge is extremely variable during individual passes of the rat through the firing field.

The idea that the rat hippocampus stores a map of space is based on the existence of "place cells" that show "location-specific" firing. The discharge of place cells is confined with remarkable precision to a cell-specific part of the environment called the cell's "firing field." We demonstrate here that firing is not nearly as reliable in the time domain as in the positional domain. Discharge during passes through the firing field was compared with a model with Poisson variance of the location-specific firing determined by the time-averaged positional firing rate distribution. Place cells characteristically fire too little or too much compared with expectations from the random model. This fundamental property of place cells is referred to as "excess firing variance" and has three main implications: (i) Place cell discharge is not only driven by the summation of many small, asynchronous excitatory synaptic inputs. (ii) Place cell discharge may encode a signal in addition to the current head location. (iii) The excess firing variance helps explain why the errors in computing the rat's position from the simultaneous activity of many place cells are large.

Variable place-cell coupling to a continuously viewed stimulus: evidence that the hippocampus acts as a perceptual system.

A key feature of perception is that the interpretation of a single, continuously available stimulus can change from time to time. This aspect of perception is well illustrated by the use of ambiguous figures that can be seen in two different ways. When people view such a stimulus they almost universally describe what they are seeing as jumping between two states. If it is agreed that this perceptual phenomenon is causally linked to the activity of nerve cells, the state jumps would have to occur in conjunction with changes in neural activity somewhere in the nervous system. Our experiments suggest that hippocampal place cells are part of a perceptual system. We conducted variations of a 'cue-card rotation' experiment on rats in which the angular position of a prominent visual stimulus on the wall of cylinder is changed in the rat's presence. The three main results are that (i) place-cell firing fields remain stationary if the cue is rotated by 180 degrees, so the relation between the cue and the field is altered; (ii) firing fields rotate by 45 degrees when the cue is rotated by 45 degrees, so the relation between the field and the card is maintained; and (iii) if the cue is first rotated by 180 degrees and then rotated in a series of 45 degrees steps, the field winds up at a different angular position relative to the card when the card is back in its original position. Thus, place cells can fire in two different ways in response to a continuously viewed stimulus. We conclude that place cells reveal that the hippocampal mapping system also has properties expected of a perceptual system.

Memory and behavior: a second generation of genetically modified mice.

The use of standard genetic techniques, such as gene targeting and transgenesis, to study cognitive function in adult animals suffers from the limitations that the gene under study is often altered in many brain regions, and that this alteration is present during the entire developmental history of the animal. Furthermore, to relate cognitive defects to neuronal mechanisms of memory, studies have relied on examining long-term potentiation - an artificially induced form of synaptic plasticity. Recent technical advances allow the expression of a genetic alteration in mice to be restricted both anatomically and temporally, making possible a more precise examination of the role of various forms of synaptic plasticity, such as long-term potentiation and long-term depression, in memory formation. Recordings from so-called 'place cells' -hippocampal cells that encode spatial location -in freely moving, genetically modified mice have further advanced our understanding of how the actual cellular representation of space is influenced by genetic alterations that affect long-term potentiation.