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Recent clinical trials of drugs enhancing cyclic guanosine monophosphate (cGMP) signaling for cardiovascular diseases have renewed interest in cGMP biology within the kidney. However, the role of cGMP signaling in glomerular endothelial cells (GECs) and podocytes remains largely unexplored. Using acute kidney slices from mice expressing the FRET-based cGMP biosensor cGi500 in endothelial cells or podocytes enabled real-time visualization of cGMP. Stimulation with atrial natriuretic peptide (ANP) or SNAP (NO donor) and various phosphodiesterase (PDE) inhibitors elevated intracellular cGMP in both cell types. GECs showed a transient cGMP response upon particulate or soluble guanylyl cyclase activation, while the cGMP response in podocytes reached a plateau following ANP administration. Co-stimulation (ANP + SNAP) led to an additive response in GECs. The administration of PDE inhibitors revealed a broader basal PDE activity in GECs dominated by PDE2a. In podocytes, basal PDE activity was mainly restricted to PDE3 and PDE5 activity. Our data demonstrate the existence of both guanylyl cyclase pathways in GECs and podocytes with cell-specific differences in cGMP synthesis and degradation, potentially suggesting new therapeutic options for kidney diseases., (© 2024. The Author(s).)

Rationale & Objective: Hyponatremia is the most common electrolyte disorder and is associated with significant morbidity and mortality. This study investigated neurocognitive impairment, brain volume, and alterations in magnetic resonance imaging (MRI)-based measures of cerebral function in patients before and after treatment for hyponatremia., Study Design: Prospective cohort study., Setting & Participants: Patients with presumed chronic hyponatremia without signs of hypo- or hypervolemia treated in the emergency department of a German tertiary-care hospital., Exposure: Hyponatremia (ie, plasma sodium concentration [Na + ]<125mmol/L) before and after treatment leading to [Na + ]>130mmol/L., Outcomes: Standardized neuropsychological testing (Mini-Mental State Examination, DemTect, Trail Making Test A/B, Beck Depression Inventory, Timed Up and Go) and resting-state MRI were performed before and after treatment of hyponatremia to assess total brain and white and gray matter volumes as well as neuronal activity and its synchronization., Analytical Approach: Changes in outcomes after treatment for hyponatremia assessed using bootstrapped confidence intervals and Cohen d statistic. Associations between parameters were assessed using correlation analyses., Results: During a 3.7-year period, 26 patients were enrolled. Complete data were available for 21 patients. Mean [Na + ]s were 118.4mmol/L before treatment and 135.5mmol/L after treatment. Most measures of cognition improved significantly. Comparison of MRI studies showed a decrease in brain tissue volumes, neuronal activity, and synchronization across all gray matter after normalization of [Na + ]. Volume effects were particularly prominent in the hippocampus. During hyponatremia, synchronization of neuronal activity was negatively correlated with [Na + ] (r=-0.836; 95% CI, -0.979 to-0.446) and cognitive function (Mini-Mental State Examination, r=-0.523; 95% CI, -0.805 to-0.069; DemTect, r=-0.744; 95% CI, -0.951 to-0.385; and Trail Making Test A, r=0.692; 95% CI, 0.255-0.922)., Limitations: Small sample size, insufficient quality of several MRI scans as a result of motion artifact., Conclusions: Resolution of hyponatremia was associated with improved cognition and reductions in brain volumes and neuronal activity. Impaired cognition during hyponatremia is closely linked to increased neuronal activity rather than to tissue volumes. Furthermore, the hippocampus appears to be particularly susceptible to hyponatremia, exhibiting pronounced changes in tissue volume., Plain-Language Summary: Hyponatremia is a common clinical problem, and patients often present with neurologic symptoms that are at least partially reversible. This study used neuropsychological testing and magnetic resonance imaging to examine patients during and after correction of hyponatremia. Treatment led to an improvement in patients' cognition as well as a decrease in their brain volumes, spontaneous neuronal activity, and synchronized neuronal activity between remote brain regions. Volume effects were particularly prominent in the hippocampus, an area of the brain that is important for the modulation of memory. During hyponatremia, patients with the lowest sodium concentrations had the highest levels of synchronized neuronal activity and the poorest cognitive test results., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

In recent years functional multiphoton (MP) imaging of vital mouse tissues and stimulation emission depletion (STED) imaging of optically cleared tissues allowed new insights into kidney biology. Here, we present a novel workflow where MP imaging of calcium signals can be combined with super-resolved STED imaging for morphological analysis of the slit diaphragm (SD) within the same glomerulus. Mice expressing the calcium indicator GCaMP3 in podocytes served as healthy controls or were challenged with two different doses of nephrotoxic serum (NTS). NTS induced glomerular damage in a dose dependent manner measured by shortening of SD length. In acute kidney slices (AKS) intracellular calcium levels increased upon disease but showed a high variation between glomeruli. We could not find a clear correlation between intracellular calcium levels and SD length in the same glomerulus. Remarkably, analysis of the SD morphology of glomeruli selected during MP calcium imaging revealed a higher percentage of completely disrupted SD architecture than estimated by STED imaging alone. Our novel co-imaging protocol is applicable to a broad range of research questions. It can be used with different tissues and is compatible with diverse reporters and target proteins., (© 2024. The Author(s).)

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 Ca 2+ -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 Ca 2+ concentration ([Ca 2+ ] i ), the experimental design was repeated in mice expressing podocyte-specific Ca 2+ sensor. MMF ameliorated the proteinuria and crescent formation induced by NTS. We identified significant changes in the abundance of proteins involved in Ca 2+ signaling and actin cytoskeleton regulation, which was further confirmed by direct [Ca 2+ ] i imaging in podocytes showing decreased Ca 2+ 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 [Ca 2+ ] 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., (© 2023. Springer Nature Limited.)

Objective: Achieving recommended targets of sodium correction is challenging to physicians treating hyponatraemia. Plasma sodium has to be increased effectively, yet overcorrection must be prevented. This is often hampered by a high variability of responses to treatment. Here, we sought to delineate factors influencing sodium evolution., Design: We retrospectively analysed 3460 patients from the multinational Hyponatraemia Registry comprising a wide range of hyponatraemia aetiologies and treatment strategies., Methods: Multivariable linear mixed effects models were applied to identify predictors of plasma sodium evolution within the first 24 h of treatment., Results: Evolution of sodium levels over time showed a curvilinear pattern with steeper rise at earlier time points. Baseline sodium showed the most pronounced impact with an additional increment of 3.12 mEq/L for every 10 mEq/L initial sodium reduction. With sodium increments of 1.9 mEq/L and 1.4 mEq/L per 24 h, respectively, the entities hypovolaemic and thiazide-associated hyponatraemia were independent factors for sodium evolution. Therapeutic regimens using hypertonic saline (4.6 mEq/L/24 h), tolvaptan (3.4 mEq/L/24 h), or combination therapy (2.6 mEq/L/24 h) were also associated with a significantly larger sodium rise when compared with no active treatment., Conclusions: Choice and dosing of active hyponatraemia therapy should be adjusted not only according to aetiology but most importantly to pretreatment sodium. Although counterintuitive, less aggressive therapy in more profound hyponatraemia might be safer but yet effective at least in less severe cases., Competing Interests: Conflicts of interest: V.B. is a consultant and his institution received research support from Otsuka for the Hyponatraemia Registry. He received travel support and fees for data review activities and served on a speakers’ bureau and advisory board panellist for Otsuka. F.G. and her institution received research support from Otsuka for participating in the Hyponatraemia Registry. All other authors declared no competing interests., (© The Author(s) 2023. Published by Oxford University Press on behalf of European Society of Endocrinology.)

Background: The current COVID-19 pandemic, despite the availability of rapid tests and the start of the vaccination campaign, continues to pose major challenges to emergency departments (ED). Structured collection of demographic, clinical, as well as treatment-related data provides the basis for establishing evidence-based processes and treatment concepts., Aim of the Work: To present the systematic collection of clinical parameters in patients with suspected COVID-19 in the Registry for COVID-19 in the Emergency Room (ReCovER) and descriptive presentation of the first 1000 patients., Materials and Methods: Data from patients with suspected COVID-19, regardless of evidence of SARS-CoV‑2 infection, are continuously entered into a web-based, anonymized registry in ED at six university hospitals., Results: Between 19 May 2020 and 13 January 2021, 1000 patients were entered into the registry, of whom 594 patients (59.4%) were in the SARS-CoV‑2 positive group (PG) and 406 patients (40.6%) were in the negative group (NG). Patients of the PG had significantly fewer pre-existing conditions and a significantly longer latency between symptom onset and presentation to the ED (median 5 vs. 3 days), were more likely to suffer from cough, myalgia, fatigue, and loss of smell/taste and had significantly higher oxygen requirements than NG patients. The rate of severe disease progression was significantly higher in the PG, and persistent symptoms were more common after discharge (11.1 vs. 4.6%)., Conclusions: The multicenter collection of comprehensive clinical data on COVID-19 suspected cases in the ED allows analysis of aspects specific to the situation in Germany in particular. This is essential for a targeted review and adaptation of internationally published strategies., (© 2021. The Author(s).)

Acute kidney injury is a frequent complication in the clinical setting and associated with significant morbidity and mortality. Preconditioning with short-term caloric restriction is highly protective against kidney injury in rodent ischemia reperfusion injury models. However, the underlying mechanisms are unknown hampering clinical translation. Here, we examined the molecular basis of caloric restriction-mediated protection to elucidate the principles of kidney stress resistance. Analysis of an RNAseq dataset after caloric restriction identified Cyp4a12a, a cytochrome exclusively expressed in male mice, to be strongly downregulated after caloric restriction. Kidney ischemia reperfusion injury robustly induced acute kidney injury in male mice and this damage could be markedly attenuated by pretreatment with caloric restriction. In females, damage was significantly less pronounced and preconditioning with caloric restriction had only little effect. Tissue concentrations of the metabolic product of Cyp4a12a, 20-hydroxyeicosatetraenoic acid (20-HETE), were found to be significantly reduced by caloric restriction. Conversely, intraperitoneal supplementation of 20-HETE in preconditioned males partly abrogated the protective potential of caloric restriction. Interestingly, this effect was accompanied by a partial reversal of caloric restriction--induced changes in protein but not RNA expression pointing towards inflammation, endoplasmic reticulum stress and lipid metabolism. Thus, our findings provide an insight into the mechanisms underlying kidney protection by caloric restriction. Hence, understanding the mediators of preconditioning is an important prerequisite for moving towards translation to the clinical setting., (Copyright © 2022 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Aim of the Study: Description and comparison of cohort characteristics and outcome of adult patients with out-of-hospital cardiac arrest (OHCA) attributed to poisoning (P-OHCA) versus patients with OHCA attributed to other medical causes (NP-OHCA)., Methods: We included all patients who received cardiopulmonary resuscitation after OHCA between January 2011 and December 2020 from German emergency medical services with good data quality in the German Resuscitation Registry., Exclusion Criteria: patients < 18 years of age or OHCA attributed to trauma, drowning, intracranial bleeding or exsanguination., Results: Patients with P-OHCA (n = 574) were significantly younger compared to NP-OHCA (n = 40,146) (median age of 43 (35-54) years vs. 73 (62-82) years; p < 0.001). Cardiac arrest in P-OHCA patients was significantly less often witnessed by bystanders (41.8 % vs. 66.2 %, p < 0.001). Asystole was the predominant initial rhythm in P-OHCA patients (73.5% vs. 53.7%, p < 0.001) while ventricular fibrillation (VF) and pulseless electrical activity (PEA) were less common (9.2% vs. 25.1% and 16.2 % vs. 20.5%, p < 0.001). P-OHCA had a higher chance of survival with good neurological outcome at hospital discharge (15.2 vs. 8.8 % p < 0.001) and poisoning was an independent protective prognostic factor in multivariate analysis (OR 2.47, 95%-CI [1.71-3.57]). P-OHCA patients with initial PEA survival with good neurological outcome was comparable to initial VF (34.3 % vs. 37.7%)., Conclusion: Patients in the P-OHCA group had a significantly higher chance of survival with good neurological outcome and PEA as initial rhythm was as favourable as initial VF. Therefore, in P-OHCA patients resuscitation efforts should be extended., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: PR is the coordinator of the GRR. JW, JTG and SSE are members of the steering committee of the GRR JW received travel grants from ZOLL medical. MH received grants or contracts from the Deutsche Forschungsgemeinschaft (DFG) and the Center for Molecular Medicine Cologne. JTG is the leader of the European Resuscitation Registry and received payment or honoraria for lectures, presentations, speakers bureaus, manuscript writing or educational events by Weinmann-Emergency, ZOLL, BARD, Fresenius, Corpuls, Braincool. All other authors have nothing to disclose., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Background: The impact of chronic moderate and profound hyponatremia on neurocognitive performance, motor skills, and mood stability has not been investigated systematically so far, and results regarding mild to moderate hyponatremia are inconsistent. Furthermore, it is not known whether treatment has an effect on outcome in these patients., Methods: A total of 130 hospitalized patients with confirmed euvolemic hyponatremia (<130 mEq/L) were subjected to a test battery (Mini-Mental State Examination, DemTect, Trail-Making Tests A and B, Beck Depression Inventory, Timed-up-and-go Test) before and after treatment; additionally, 50 normonatremic group-matched patients served as reference group., Results: The scores of all tested domains were significantly worse in the hyponatremia group (median serum sodium [Na + ] 122 (119-126) mEq/L) as compared to the reference group (P <0.001), and the odds of obtaining a pathological test result increased markedly with more profound hyponatremic states (odds ratios between 5.0 and 21.8 in the group with Na + <120 mEq/L compared to reference group). Inversely, treatment led to a significant amelioration of all test results with medium to large effect sizes. Linear regression models revealed the increment of Na + as an important predictor of test outcome., Conclusion: We demonstrate a clear association between lower levels of Na + beyond mild hyponatremia and impairment of neurocognitive and motor performance as well as mood disorders. Our analysis further suggests a causal role of hyponatremia in this context. However, there are apparent differences between the distinct tested domains warranting further investigations., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Mammalian kidneys constantly filter large amounts of liquid, with almost complete retention of albumin and other macromolecules in the plasma. Breakdown of the three-layered renal filtration barrier results in loss of albumin into urine (albuminuria) across the wall of small renal capillaries, and is a leading cause of chronic kidney disease. However, exactly how the renal filter works and why its permeability is altered in kidney diseases is poorly understood. Here we show that the permeability of the renal filter is modulated through compression of the capillary wall. We collect morphometric data prior to and after onset of albuminuria in a mouse model equivalent to a human genetic disease affecting the renal filtration barrier. Combining quantitative analyses with mathematical modelling, we demonstrate that morphological alterations of the glomerular filtration barrier lead to reduced compressive forces that counteract filtration pressure, thereby resulting in capillary dilatation, and ultimately albuminuria. Our results reveal distinct functions of the different layers of the filtration barrier and expand the molecular understanding of defective renal filtration in chronic kidney disease.

Background: Inhibition of angiotensin II (AngII) signaling, a therapeutic mainstay of glomerular kidney diseases, is thought to act primarily through regulating glomerular blood flow and reducing filtration pressure. Although extravascular actions of AngII have been suggested, a direct effect of AngII on podocytes has not been demonstrated in vivo ., Methods: To study the effects of AngII on podocyte calcium levels in vivo , we used intravital microscopy of the kidney in mice expressing the calcium indicator protein GCaMP3., Results: In healthy animals, podocytes displayed limited responsiveness to AngII stimulation. In contrast, in animals subjected to either adriamycin-induced acute chemical injury or genetic deletion of the podocin-encoding gene Nphs2 , the consequent podocyte damage and proteinuria rendered the cells responsive to AngII and resulted in AngII-induced calcium transients in significantly more podocytes. The angiotensin type 1 receptor blocker losartan could fully inhibit this response. Also, responsiveness to AngII was at least partly mediated through the transient receptor potential channel 6, which has been implicated in podocyte calcium handling. Interestingly, loss of a single Nphs2 allele also increased podocytes' responsiveness to AngII signaling. This direct effect of AngII on injured podocytes results in increased calcium transients, which can further aggravate the underlying kidney disease., Conclusions: Our discovery that podocytes become sensitized to AngII-induced calcium signaling upon injury might explain results from large, randomized, controlled trials in which improved renal outcomes occur only in the subgroup of patients with proteinuria, indicating podocyte damage. Our findings also emphasize the need to treat every patient with a glomerular disease with either an angiotensin-converting enzyme inhibitor or an angiotensin type 1 receptor blocker., (Copyright © 2020 by the American Society of Nephrology.)

In diseases of many parenchymatous organs, heterogeneous deterioration of individual functional units determines the clinical prognosis. However, the molecular characterization at the level of such individual subunits remains a technological challenge that needs to be addressed in order to better understand pathological mechanisms. Proteinuric glomerular kidney diseases are frequent and assorted diseases affecting a fraction of glomeruli and their draining tubules to variable extents, and for which no specific treatment exists. Here, we developed and applied a mass spectrometry-based methodology to investigate heterogeneity of proteomes from individually isolated nephron segments from mice with proteinuric kidney disease. In single glomeruli from two different mouse models of sclerotic glomerular disease, we identified a coherent protein expression module consisting of extracellular matrix protein deposition (reflecting glomerular sclerosis), glomerular albumin (reflecting proteinuria) and LAMP1, a lysosomal protein. This module was associated with a loss of podocyte marker proteins while genetic ablation of LAMP1-correlated lysosomal proteases could ameliorate glomerular damage in vivo. Furthermore, proteomic analyses of individual glomeruli from patients with genetic sclerotic and non-sclerotic proteinuric diseases revealed increased abundance of lysosomal proteins, in combination with a decreased abundance of mutated gene products. Thus, altered protein homeostasis (proteostasis) is a conserved key mechanism in proteinuric kidney diseases. Moreover, our technology can capture intra-individual variability in diseases of the kidney and other tissues at a sub-biopsy scale., (Copyright © 2018 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Chronic alterations in calcium (Ca 2+ ) signalling in podocytes have been shown to cause proteinuria and progressive glomerular diseases. However, it is unclear whether short Ca 2+ peaks influence glomerular biology and cause podocyte injury. Here we generated a DREADD (Designer Receptor Exclusively Activated by a Designer Drug) knock-in mouse line to manipulate intracellular Ca 2+ levels. By mating to a podocyte-specific Cre driver we are able to investigate the impact of Ca 2+ peaks on podocyte biology in living animals. Activation of the engineered G-protein coupled receptor with the synthetic compound clozapine-N-oxide (CNO) evoked a short and transient Ca 2+ peak in podocytes immediately after CNO administration in vivo. Interestingly, this Ca 2+ peak did neither affect glomerular perfusion nor filtration in the animals. Moreover, no obvious alterations in the glomerular morphology could be observed. Taken together, these in vivo findings suggest that chronic alterations and calcium overload rather than an induction of transient Ca 2+ peaks contribute to podocyte disease.

Intracellular calcium ([Ca²⁺]i) signaling mediates physiological and pathological processes in multiple organs, including the renal podocyte; however, in vivo podocyte [Ca²⁺]i dynamics are not fully understood. Here we developed an imaging approach that uses multiphoton microscopy (MPM) to directly visualize podocyte [Ca²⁺]i dynamics within the intact kidneys of live mice expressing a fluorescent calcium indicator only in these cells. [Ca²⁺]i was at a low steady-state level in control podocytes, while Ang II infusion caused a minor elevation. Experimental focal podocyte injury triggered a robust and sustained elevation of podocyte [Ca²⁺]i around the injury site and promoted cell-to-cell propagating podocyte [Ca²⁺]i waves along capillary loops. [Ca²⁺]i wave propagation was ameliorated by inhibitors of purinergic [Ca²⁺]i signaling as well as in animals lacking the P2Y2 purinergic receptor. Increased podocyte [Ca²⁺]i resulted in contraction of the glomerular tuft and increased capillary albumin permeability. In preclinical models of renal fibrosis and glomerulosclerosis, high podocyte [Ca²⁺]i correlated with increased cell motility. Our findings provide a visual demonstration of the in vivo importance of podocyte [Ca²⁺]i in glomerular pathology and suggest that purinergic [Ca²⁺]i signaling is a robust and key pathogenic mechanism in podocyte injury. This in vivo imaging approach will allow future detailed investigation of the molecular and cellular mechanisms of glomerular disease in the intact living kidney.

Background: Various cell types, including podocytes and parietal epithelial cells, play important roles in the development and progression of glomerular kidney diseases, albuminuria, and glomerulosclerosis. Besides their role in renal pathologies, glomerular cells have emerging new functions in endogenous repair mechanisms. A better understanding of the dynamics of the glomerular environment and cellular composition in an intact living kidney is critically important for the development of new regenerative therapeutic strategies for kidney diseases. However, progress in this field has been hampered by the lack of in vivo research tools., Summary: This review summarizes the current state-of-the-art in the application of the unique intravital imaging technology of multiphoton fluorescence microscopy for the dynamic visualization of glomerular structure and function over time in the intact, living kidney. Recently, this imaging approach in combination with transgenic mouse models allowed tracking of the fate of individual glomerular cells in vivo over several days and depicted the highly dynamic nature of the glomerular environment, particularly in disease conditions., Key Messages: The technology is ready and available for future intravital imaging studies investigating new glomerular regenerative approaches in animal models.

Podocytes are critical in the maintenance of a healthy glomerular filter; however, they have been difficult to study in the intact kidney because of technical limitations. Here we report the development of serial multiphoton microscopy (MPM) of the same glomeruli over several days to visualize the motility of podocytes and parietal epithelial cells (PECs) in vivo. In podocin-GFP mice, podocytes formed sporadic multicellular clusters after unilateral ureteral ligation and migrated into the parietal Bowman's capsule. The tracking of single cells in podocin-confetti mice featuring cell-specific expression of CFP, GFP, YFP or RFP revealed the simultaneous migration of multiple podocytes. In phosphoenolpyruvate carboxykinase (PEPCK)-GFP mice, serial MPM found PEC-to-podocyte migration and nanotubule connections. Our data support a highly dynamic rather than a static nature of the glomerular environment and cellular composition. Future application of this new approach should advance our understanding of the mechanisms of glomerular injury and regeneration.

Transplantation is invariably associated with ischemia-reperfusion injury (IRI), inflammation and rejection. Resultant cell death has morphological features of necrosis but programmed cell death has been synonymous with apoptosis until pathways of regulated necrosis (RN) have been described. The best-studied RN pathway, necroptosis, is triggered by perturbation of caspase-8-mediated apoptosis and depends on receptor-interacting protein kinases 1 and 3 (RIPK1/RIPK3) as well as mixed linage kinase domain like to form the necroptosome. The release of cytosolic content and cell death-associated molecular patterns (CDAMPs) can trigger innate and promote adaptive immune responses. Thus, the form of cell death can substantially influence alloimmunity and graft survival. Necroptosis is a key element of IRI, and RIPK1 interference by RN-specific inhibitors such as necrostatin-1 protects from IRI in kidney, heart and brain. Necroptosis may be a general mechanism in response to other forms of inflammatory organ injury, and will likely emerge as a promising target in solid organ transplantation. As second-generation RIPK1 and RIPK3 inhibitors become available, clinical trials for the prevention of delayed graft function and attenuation of allograft rejection-mediated injury will emerge. These efforts will accelerate upon further identification of critical necroptosis-triggering receptor(s)., (© Copyright 2013 The American Society of Transplantation and the American Society of Transplant Surgeons.)

In this review, we highlight the major scientific breakthroughs in kidney research achieved using multiphoton microscopy (MPM) and summarize the milestones in the technological development of kidney MPM during the past 10 years. Since more and more renal laboratories invest in MPM worldwide, we discuss future directions and provide practical, useful tips and examples for the application of this still-emerging optical sectioning technology. Advantages of using MPM in various kidney preparations that range from freshly dissected individual glomeruli or the whole kidney in vitro to MPM of the intact mouse and rat kidney in vivo are reviewed. Potential combinations of MPM with micromanipulation techniques including microperfusion and micropuncture are also included. However, we emphasize the most advanced and complex, quantitative in vivo imaging applications as the ultimate use of MPM since the true mandate of this technology is to look inside intact organs in live animals and humans.