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Tissue regeneration is a process that recapitulates and restores organ structure and function. Although previous studies have demonstrated wound-induced hair neogenesis (WIHN) in laboratory mice (Mus), the regeneration is limited to the center of the wound unlike those observed in African spiny (Acomys) mice. Tissue mechanics have been implicated as an integral part of tissue morphogenesis. Here, we use the WIHN model to investigate the mechanical and molecular responses of laboratory and African spiny mice, and report these models demonstrate opposing trends in spatiotemporal morphogenetic field formation with association to wound stiffness landscapes. Transcriptome analysis and K14-Cre-Twist1 transgenic mice show the Twist1 pathway acts as a mediator for both epidermal-dermal interactions and a competence factor for periodic patterning, differing from those used in development. We propose a Turing model based on tissue stiffness that supports a two-scale tissue mechanics process: (1) establishing a morphogenetic field within the wound bed (mm scale) and (2) symmetry breaking of the epidermis and forming periodically arranged hair primordia within the morphogenetic field (μm scale). Thus, we delineate distinct chemo-mechanical events in building a Turing morphogenesis-competent field during WIHN of laboratory and African spiny mice and identify its evo-devo advantages with perspectives for regenerative medicine.

BACKGROUND: The endocytic reabsorption of proteins in the proximal tubule requires a complex machinery and defects can lead to tubular proteinuria. The precise mechanisms of endocytosis and processing of receptors and cargo are incompletely understood. EHD1 belongs to a family of proteins presumably involved in the scission of intracellular vesicles and in ciliogenesis. However, the relevance of EHD1 in human tissues, in particular in the kidney, was unknown.METHODS: Genetic techniques were used in patients with tubular proteinuria and deafness to identify the disease-causing gene. Diagnostic and functional studies were performed in patients and disease models to investigate the pathophysiology.RESULTS: We identified six individuals (5-33 years) with proteinuria and a high-frequency hearing deficit associated with the homozygous missense variant c.1192C>T (p.R398W) in EHD1. Proteinuria (0.7-2.1 g/d) consisted predominantly of low molecular weight proteins, reflecting impaired renal proximal tubular endocytosis of filtered proteins. Ehd1 knockout and Ehd1R398W/R398W knockin mice also showed a high-frequency hearing deficit and impaired receptor-mediated endocytosis in proximal tubules, and a zebrafish model showed impaired ability to reabsorb low molecular weight dextran. Interestingly, ciliogenesis appeared unaffected in patients and mouse models. In silico structural analysis predicted a destabilizing effect of the R398W variant and possible inference with nucleotide binding leading to impaired EHD1 oligomerization and membrane remodeling ability.CONCLUSIONS: A homozygous missense variant of EHD1 causes a previously unrecognized autosomal recessive disorder characterized by sensorineural deafness and tubular proteinuria. Recessive EHD1 variants should be considered in individuals with hearing impairment, especially if tubular proteinuria is noted.

Endothelial cells are important in the maintenance of healthy blood vessels and in the development of vascular diseases. However, the origin and dynamics of endothelial precursors and remodeling at the single-cell level have been difficult to study in vivo owing to technical limitations. Therefore, we aimed to develop a direct visual approach to track the fate and function of single endothelial cells over several days and weeks in the same vascular bed in vivo using multiphoton microscopy (MPM) of transgenic Cdh5-Confetti mice and the kidney glomerulus as a model. Individual cells of the vascular endothelial lineage were identified and tracked owing to their unique color combination, based on the random expression of cyan/green/yellow/red fluorescent proteins. Experimental hypertension, hyperglycemia, and laser-induced endothelial cell ablation rapidly increased the number of new glomerular endothelial cells that appeared in clusters of the same color, suggesting clonal cell remodeling by local precursors at the vascular pole. Furthermore, intravital MPM allowed the detection of distinct structural and functional alterations of proliferating endothelial cells. No circulating Cdh5-Confetti+ cells were found in the renal cortex. Moreover, the heart, lung, and kidneys showed more significant clonal endothelial cell expansion compared with the brain, pancreas, liver, and spleen. In summary, we have demonstrated that serial MPM of Cdh5-Confetti mice in vivo is a powerful technical advance to study endothelial remodeling and repair in the kidney and other organs under physiological and disease conditions.

Acute kidney injury (AKI) is associated with an increased risk of CKD. Injury-induced multifaceted renal cell-to-cell crosstalk can either lead to successful self-repair or chronic fibrosis and inflammation. In this mini-review, we will discuss critical renal cell types acting as victims or executioners in AKI pathology and introduce intravital imaging as a powerful technique to further dissect these cell-to-cell interactions.

Lupus nephritis (LN) is a major organ complication and cause of morbidity and mortality in patients with systemic lupus erythematosus (SLE). There is an unmet medical need for developing more efficient and specific, mechanism-based therapies, which depends on improved understanding of the underlying LN pathogenesis. Here we present direct visual evidence from high-power intravital imaging of the local kidney tissue microenvironment in mouse models showing that activated memory T cells originated in immune organs and the LN-specific robust accumulation of the glomerular endothelial glycocalyx played central roles in LN development. The glomerular homing of T cells was mediated via the direct binding of their CD44 to the hyaluronic acid (HA) component of the endothelial glycocalyx, and glycocalyx-degrading enzymes efficiently disrupted homing. Short-course treatment with either hyaluronidase or heparinase III provided long-term organ protection as evidenced by vastly improved albuminuria and survival rate. This glycocalyx/HA/memory T cell interaction is present in multiple SLE-affected organs and may be therapeutically targeted for SLE complications, including LN., A combined immunology and renal pathophysiology study of the local kidney tissue microenvironment in lupus identifies a key role of glomerular endothelial glycocalyx in disease development.

Intravital multiphoton microscopy of the kidney is a powerful technique to study alterations in tissue morphology and function simultaneously in the living animal and represents a dynamic and developing research tool in the field. Recent technological advances include serial intravital multiphoton microscopy of the same kidney regions over several weeks and combined with ex vivo histology for cellular biomarker expression of the same cells, which had been subject to serial imaging before. Thus, serial intravital multiphoton microscopy followed by ex vivo histology provides unique tools to perform long-term cell fate tracing of the same renal cells during physiological and pathophysiological conditions, thereby allowing the detection of structural changes of the same renal cells over time. Examples include renal cell migration and proliferation while linking these events to local functional alterations and eventually to the expression of distinct cellular biomarkers. Here, we provide a detailed step-by-step protocol to facilitate serial intravital multiphoton microscopy for long-term in vivo tracking of renal cells and subsequent ex vivo histology for immunohistological staining of the same cells in the fixed tissue.

Background The kidney is considered to be a structurally stable organ with limited baseline cellular turnover. Nevertheless, single cells must be constantly replaced to conserve the functional integrity of the organ. PDGF chain B (PDGF-BB) signaling through fibroblast PDGF receptor- β (PDGFR β ) contributes to interstitial-epithelial cell communication and facilitates regenerative functions in several organs. However, the potential role of interstitial cells in renal tubular regeneration has not been examined. Methods In mice with fluorescent protein expression in renal tubular cells and PDGFR β -positive interstitial cells, we ablated single tubular cells by high laser exposure. We then used serial intravital multiphoton microscopy with subsequent three-dimensional reconstruction and ex vivo histology to evaluate the cellular and molecular processes involved in tubular regeneration. Results Single-tubular cell ablation caused the migration and division of dedifferentiated tubular epithelial cells that preceded tubular regeneration. Moreover, tubular cell ablation caused immediate calcium responses in adjacent PDGFR β -positive interstitial cells and the rapid migration thereof toward the injury. These PDGFR β -positive cells enclosed the injured epithelium before the onset of tubular cell dedifferentiation, and the later withdrawal of these PDGFR β -positive cells correlated with signs of tubular cell redifferentiation. Intraperitoneal administration of trapidil to block PDGFR β impeded PDGFR β -positive cell migration to the tubular injury site and compromised the recovery of tubular function. Conclusions Ablated tubular cells are exclusively replaced by resident tubular cell proliferation in a process dependent on PDGFR β -mediated communication between the renal interstitium and the tubular system.

The NPHS2 gene, encoding the slit diaphragm protein podocin, accounts for genetic and sporadic forms of nephrotic syndrome (NS). Patients with NS often present symptoms of volume retention, such as oedema formation or hypertension. The primary dysregulation in sodium handling involves an inappropriate activation of the epithelial sodium channel, ENaC. Plasma proteases in a proteinuria‐dependent fashion have been made responsible; however, referring to the timeline of symptoms occurring and underlying mechanisms, contradictory results have been published. Characterizing the mouse model of podocyte inactivation of NPHS2 (Nphs2∆pod) with respect to volume handling and proteinuria revealed that sodium retention, hypertension and gross proteinuria appeared sequentially in a chronological order. Detailed analysis of Nphs2∆pod during early sodium retention, revealed increased expression of full‐length ENaC subunits and αENaC cleavage product with concomitant increase in ENaC activity as tested by amiloride application, and augmented collecting duct Na+/K+‐ATPase expression. Urinary proteolytic activity was increased and several proteases were identified by mass spectrometry including cathepsin B, which was found to process αENaC. Renal expression levels of precursor and active cathepsin B were increased and could be localized to glomeruli and intercalated cells. Inhibition of cathepsin B prevented hypertension. With the appearance of gross proteinuria, plasmin occurs in the urine and additional cleavage of γENaC is encountered. In conclusion, characterizing the volume handling of Nphs2∆pod revealed early sodium retention occurring independent to aberrantly filtered plasma proteases. As an underlying mechanism cathepsin B induced αENaC processing leading to augmented channel activity and hypertension was identified.

Intravital multiphoton microscopy of the kidney is a powerful technique to study alterations in tissue morphology and function simultaneously in the living animal and represents a dynamic and developing research tool in the field. Recent technological advances include serial intravital multiphoton microscopy of the same kidney regions over several weeks and combined with ex vivo histology for cellular biomarker expression of the same cells, which had been subject to serial imaging before. Thus, serial intravital multiphoton microscopy followed by ex vivo histology provides unique tools to perform long-term cell fate tracing of the same renal cells during physiological and pathophysiological conditions, thereby allowing the detection of structural changes of the same renal cells over time. Examples include renal cell migration and proliferation while linking these events to local functional alterations and eventually to the expression of distinct cellular biomarkers. Here, we provide a detailed step-by-step protocol to facilitate serial intravital multiphoton microscopy for long-term in vivo tracking of renal cells and subsequent ex vivo histology for immunohistological staining of the same cells in the fixed tissue.

Fluorescence microscopy techniques are powerful tools to study tissue dynamics, cellular function and biology both in vivo and in vitro. These tools allow for functional assessment and quantification along with qualitative analysis, thus providing a comprehensive understanding of various cellular processes under normal physiological and disease conditions. The main focus of this chapter is the recently developed method of serial intravital multiphoton microscopy that has helped shed light on the dynamic alterations of the spatial distribution and fate of single renal cells or cell populations and their migration patterns in the same tissue region over several days in response to various stimuli within the living kidney. This technique is very useful for studying in vivo the molecular and cellular mechanisms of tissue remodeling and repair after injury. In addition, complementary in vitro imaging tools are also described and discussed, like tissue clearing techniques and protein synthesis measurement in tissues in situ that provide an in depth assessment of changes at the cellular level. Thus, these novel fluorescence techniques can be effectively leveraged for different tissue types, experimental conditions as well as disease models to improve our understanding of renal cell biology.

Albuminuria is a hallmark of kidney disease of various etiologies and usually caused by deterioration of glomerular filtration barrier integrity. We recently showed that angiotensin II (Ang II) acutely increases albumin filtration in the healthy kidney. Here, we used intravital microscopy to assess the effects of Ang II on podocyte function in rats. Acute infusion of 30, 60, or 80 ng/kg per minute Ang II enhanced the endocytosis of albumin by activation of the type 1 Ang II receptor and resulted in an average (±SEM) of 3.7±2.2, 72.3±18.6 (Pµm³ (P

To elucidate the physiologic function of renal globotriaosylceramide (Gb3/CD77), which up-to-date has been associated exclusively with Shiga toxin binding, we have analyzed renal function in Gb3-deficient mice. Gb3 synthase KO (Gb3S−/−) mice displayed an increased renal albumin and low molecular weight protein excretion compared to WT. Gb3 localized at the brush border and within vesicular structures in WT proximal tubules and has now been shown to be closely associated with the receptor complex megalin/cubilin and with albumin uptake. In two clinically relevant mouse models of acute kidney injury caused by myoglobin as seen in rhabdomyolysis and the aminoglycoside gentamicin, Gb3S−/− mice showed a preserved renal function and morphology, compared to WT. Pharmacologic inhibition of glucosylceramide-based glycosphingolipids, including Gb3, in WT mice corroborated the results of genetically Gb3-deficient mice. In conclusion, our data significantly advance the current knowledge on the physiologic and pathophysiologic role of Gb3 in proximal tubules, showing an involvement in the reabsorption of filtered albumin, myoglobin and the aminoglycoside gentamicin.

Receptor-interacting protein kinases 1 and 3 (RIPK1/3) have best been described for their role in mediating a regulated form of necrosis, referred to as necroptosis. During this process, RIPK3 phosphorylates mixed lineage kinase domain-like (MLKL) to cause plasma membrane rupture. RIPK3-deficient mice have recently been demonstrated to be protected in a series of disease models, but direct evidence for activation of necroptosis in vivo is still limited. Here, we sought to further examine the activation of necroptosis in kidney ischemia-reperfusion injury (IRI) and from TNFα-induced severe inflammatory response syndrome (SIRS), two models of RIPK3-dependent injury. In both models, MLKL-ko mice were significantly protected from injury to a degree that was slightly, but statistically significantly exceeding that of RIPK3-deficient mice. We also demonstrated, for the first time, accumulation of pMLKL in the necrotic tubules of human patients with acute kidney injury. However, our data also uncovered unexpected elevation of blood flow in MLKL-ko animals, which may be relevant to IRI and should be considered in the future. To further understand the mode of regulation of cell death by MLKL, we screened a panel of clinical plasma membrane channel blockers and we found phenytoin to inhibit necroptosis. However, we further found that phenytoin attenuated RIPK1 kinase activity in vitro, likely due to the hydantoin scaffold also present in necrostatin-1, and blocked upstream necrosome formation steps in the cells undergoing necroptosis. We further report that this clinically used anti-convulsant drug displayed protection from kidney IRI and TNFα-induces SIRS in vivo. Overall, our data reveal the relevance of RIPK3-pMLKL regulation for acute kidney injury and identifies an FDA-approved drug that may be useful for immediate clinical evaluation of inhibition of pro-death RIPK1/RIPK3 activities in human diseases.

Receptor-interacting protein kinases 1 and 3 (RIPK1/3) have best been described for their role in mediating a regulated form of necrosis, referred to as necroptosis. During this process, RIPK3 phosphorylates mixed lineage kinase domain-like (MLKL) to cause plasma membrane rupture. RIPK3-deficient mice have recently been demonstrated to be protected in a series of disease models, but direct evidence for activation of necroptosis in vivo is still limited. Here, we sought to further examine the activation of necroptosis in kidney ischemia-reperfusion injury (IRI) and from TNFα-induced severe inflammatory response syndrome (SIRS), two models of RIPK3-dependent injury. In both models, MLKL-ko mice were significantly protected from injury to a degree that was slightly, but statistically significantly exceeding that of RIPK3-deficient mice. We also demonstrated, for the first time, accumulation of pMLKL in the necrotic tubules of human patients with acute kidney injury. However, our data also uncovered unexpected elevation of blood flow in MLKL-ko animals, which may be relevant to IRI and should be considered in the future. To further understand the mode of regulation of cell death by MLKL, we screened a panel of clinical plasma membrane channel blockers and we found phenytoin to inhibit necroptosis. However, we further found that phenytoin attenuated RIPK1 kinase activity in vitro, likely due to the hydantoin scaffold also present in necrostatin-1, and blocked upstream necrosome formation steps in the cells undergoing necroptosis. We further report that this clinically used anti-convulsant drug displayed protection from kidney IRI and TNFα-induces SIRS in vivo. Overall, our data reveal the relevance of RIPK3-pMLKL regulation for acute kidney injury and identifies an FDA-approved drug that may be useful for immediate clinical evaluation of inhibition of pro-death RIPK1/RIPK3 activities in human diseases.

The kidney is considered to be a structurally stable organ with limited baseline cellular turnover. Nevertheless, single cells must be constantly replaced to conserve the functional integrity of the organ. PDGF chain B (PDGF-BB) signaling through fibroblast PDGF receptor-In mice with fluorescent protein expression in renal tubular cells and PDGFRSingle-tubular cell ablation caused the migration and division of dedifferentiated tubular epithelial cells that preceded tubular regeneration. Moreover, tubular cell ablation caused immediate calcium responses in adjacent PDGFRAblated tubular cells are exclusively replaced by resident tubular cell proliferation in a process dependent on PDGFR

The Na-K-2Cl cotransporter (NKCC2; BSC1) is located in the apical membrane of the epithelial cells of the thick ascending limb of the loop of Henle (TAL). NKCC2 facilitates ∼20–25% of the reuptake of the total filtered NaCl load. NKCC2 is therefore one of the transport proteins with the highest overall reabsorptive capacity in the kidney. Consequently, even subtle changes in NKCC2 transport activity considerably alter the renal reabsorptive capacity for NaCl and eventually lead to perturbations of the salt and water homoeostasis. In addition to facilitating the bulk reabsorption of NaCl in the TAL, NKCC2 transport activity in the macula densa cells of the TAL constitutes the initial step of the tubular-vascular communication within the juxtaglomerular apparatus (JGA); this communications allows the TAL to modulate the preglomerular resistance of the afferent arteriole and the renin secretion from the granular cells of the JGA. This review provides an overview of our current knowledge with respect to the general functions of NKCC2, the modulation of its transport activity by different regulatory mechanisms, and new developments in the pathophysiology of NKCC2-dependent renal NaCl transport.

Kidney cell death plays a key role in the progression of life-threatening renal diseases, such as acute kidney injury and chronic kidney disease. Injured and dying epithelial and endothelial cells take part in complex communication with the innate immune system, which drives the progression of cell death and the decrease in renal function. To improve our understanding of kidney cell death dynamics and its impact on renal disease, a study approach is needed that facilitates the visualization of renal function and morphology in real time. Intravital multiphoton microscopy of the kidney has been used for more than a decade and made substantial contributions to our understanding of kidney physiology and pathophysiology. It is a unique tool that relates renal structure and function in a time- and spatial-dependent manner. Basic renal function, such as microvascular blood flow regulation and glomerular filtration, can be determined in real time and homeostatic alterations, which are linked inevitably to cell death and can be depicted down to the subcellular level. This review provides an overview of the available techniques to study kidney dysfunction and inflammation in terms of cell death in vivo, and addresses how this novel approach can be used to improve our understanding of cell death dynamics in renal disease.

Glomerular hyperfiltration, as seen for instance in diabetic nephropathy or in response to loss of kidney tissue, induces hypertrophy of proximal tubular cells. In our study we tested whether the i...

Angiotensin-converting enzyme (ACE) inhibitors are commonly used antiproteinuric drugs. Here we assessed the effect of the ACE inhibitor enalapril on the glomerular sieving coefficient of albumin (GSCA) using intravital multiphoton microscopy. Munich Wistar Fromter (MWF) rats were used as a model of hypertension-related glomerular lesions. Young (9-week-old) MWF rats were nonproteinuric, similar to what was observed in control Wistar rats. However, urinary albumin excretion in the MWF rats gradually increased during aging, averaging 0.00062 ± 0.0001 at age 9 weeks and 0.0054 ± 0.0003 (mg/mOsmol per liter) at age 52 weeks (P < 0.0001). Albuminuria in aged MWF rats was accompanied by structural changes, which were indicative of glomerular lesions. The GSCA was low in young MWF rats but increased markedly during aging, averaging 0.00057 ± 4.7 × 10(-5) (n = 25) in young MWF rats and 0.0027 ± 0.00036 in 52-week-old MWF rats (n = 36; P < 0.0001). Treatment of proteinuric 12-month-old MWF rats with enalapril over a 4-week period reduced the GSCA from 0.0027 ± 0.00036 to 0.00139 ± 0.00013 (P = 0.0005). Similarly, urinary albumin excretion was reduced, averaging 0.0051 ± 0.0003 and 0.0036 ± 0.0005 mg/mOsmol per liter before and after enalapril administration, respectively (P = 0.0089). In parallel, enalapril treatment reduced the mean arterial blood pressure (144.6 ± 6.5 mm Hg in untreated versus 110.9 ± 0.6 mm Hg in enalapril-treated MWF rats) and increased the glomerular filtration rate from 1.64 ± 0.3 ml/min to 3.58 ± 0.3 ml/min (P = 0.0025 versus baseline). In summary, enalapril reduced the GSCA in proteinuric MWF rats, which was paralleled by a similar reduction in urinary albumin excretion. These data suggest that glomerular rather than tubular mechanisms account for the beneficial antiproteinuric effects of the ACE inhibitor.

In this study, we assessed the acute effects of angiotensin II on the albumin glomerular sieving coefficient (GSC) using intravital microscopy. The experiments were performed on Munich Wistar Froemter (MWF) rats. Alexa-Fluor-594 albumin was injected intravenously, and the fluorescence intensity in the glomerular capillaries and Bowman's space was determined to calculate the albumin GSC. The GSC was measured before and during the constant infusion of angiotensin II (10 ng·min−1·kg−1body wt). Baseline mean arterial pressure (MAP) was 99 ± 5 mmHg and stabilized at 137 ± 5 mmHg during angiotensin II infusion. The baseline GSC averaged 0.00044 ± 4.8 × 10−5and increased by 286 ± 44% after angiotensin II infusion ( P < 0.0001). The proximal tubular Alexa-Fluor-594 albumin uptake was enhanced during angiotensin II infusion (518% of the baseline value during angiotensin II vs. 218% in controls; P < 0.0001). No change in GSC was observed when the AT1antagonist losartan was injected before the start of angiotensin II infusion. The AT2antagonist PD123319 increased the baseline GSC from 0.00052 ± 3.6 × 10−5to 0.00074 ± 8.2 × 10−5( P = 0.02) without altering the MAP. During angiotensin II infusion with losartan, PD123319 increased the albumin GSC from 0.00037 ± 5.8 × 10−5to 0.00115 ± 0.00015 ( P = 0.001). When the renal perfusion pressure was mechanically controlled, the GSC increased from 0.0007 ± 0.00019 to 0.0025 ± 0.00063 during angiotensin II infusion ( P = 0.047), similar to what was observed when the renal perfusion pressure was allowed to increase. In summary, AT1activation acutely increases the albumin GSC. This effect appears to be largely independent of changes in the renal perfusion pressure. The AT2receptor partially attenuates the proteinuric effects of the AT1receptor.

Participation of connexin 40 (Cx40) in the regulation of renin secretion and in the tubuloglomerular feedback (TGF) component of renal autoregulation suggests that gap junctional coupling through Cx40 contributes to the function of the juxtaglomerular apparatus. In the present experiments, we determined the effect of targeted Cx40 deletion in C57BL/6 and FVB mice on TGF responsiveness. In C57BL/6 mice, stop-flow pressure (PSF) fell from 40.3 ± 2 to 34.5 ± 2 mmHg in wild-type (WT) and from 31 ± 1.06 to 26.6 ± 0.98 mmHg in Cx40−/− mice. PSF changes of 5.85 ± 0.67 mmHg in WT and of 4.3 ± 0.55 mmHg in Cx40−/− mice were not significantly different ( P = 0.08). In FVB mice, PSF fell from 37.4 ± 1.5 to 31.6 ± 1.5 mmHg in WT and from 28.1 ± 1.6 to 25.4 ± 1.7 mmHg in Cx40−/−, with mean TGF responses being significantly greater in WT than Cx40−/− (5.5 ± 0.55 vs. 2.7 ± 0.84 mmHg; P = 0.002). In both genetic backgrounds, PSF values were significantly lower in Cx40−/− than WT mice at all flow rates. Arterial blood pressure in the animals prepared for micropuncture was not different between WT and Cx40−/− mice. We conclude that the TGF response magnitude in superficial cortical nephrons is reduced by 30–50% in mice without Cx40, but that with the exception of a small number of nephrons, residual TGF activity is maintained. Thus gap junctional coupling appears to modulate TGF, perhaps by determining the kinetics of signal transmission.