Determining individual glomerular proteinuria and periglomerular infiltration in a cleared murine kidney by a 3-dimensional fast marching algorithm.

Three-dimensional (3D) imaging has advanced basic research and clinical medicine. However, limited resolution and imperfections of real-world 3D image material often preclude algorithmic image analysis. Here, we present a methodologic framework for such imaging and analysis for functional and spatial relations in experimental nephritis. First, optical tissue-clearing protocols were optimized to preserve fluorescence signals for light sheet fluorescence microscopy and compensated attenuation effects using adjustable 3D correction fields. Next, we adapted the fast marching algorithm to conduct backtracking in 3D environments and developed a tool to determine local concentrations of extractable objects. As a proof-of-concept application, we used this framework to determine in a glomerulonephritis model the individual proteinuria and periglomerular immune cell infiltration for all glomeruli of half a mouse kidney. A correlation between these parameters surprisingly did not support the intuitional assumption that the most inflamed glomeruli are the most proteinuric. Instead, the spatial density of adjacent glomeruli positively correlated with the proteinuria of a given glomerulus. Because proteinuric glomeruli appear clustered, this suggests that the exact location of a kidney biopsy may affect the observed severity of glomerular damage. Thus, our algorithmic pipeline described here allows analysis of various parameters of various organs composed of functional subunits, such as the kidney, and can theoretically be adapted to processing other image modalities.

The multifaceted role of the renal mononuclear phagocyte system.

The kidney contains a large and complex network of mononuclear phagocytes, which includes dendritic cells (DCs) and macrophages (MØs). The distinction between these cell types is traditionally based on the expression of molecular markers and morphology. However, several classification systems are used in parallel to identify DCs and MØs, leading to considerable uncertainty about their identity and functional roles. The discovery that a substantial proportion of macrophages in tissues like the kidney are embryonically derived further complicates the situation. Recent studies have used newly identified transcription factors such as ZBTB46 and lineage tracing techniques for classifying mononuclear phagocytes. These approaches have shed new light on the functional specialization of these cells in health and disease, uncovered an influence of the renal microenvironment and revealed considerable cellular plasticity, especially in inflammatory situations. In this review, the current knowledge about the developmental origins and versatile functional roles of DCs and MØs in kidney homeostasis and disease is discussed.

Opposing Roles of Dendritic Cell Subsets in Experimental GN.

Dendritic cells (DCs) are thought to form a dendritic network across barrier surfaces and throughout organs, including the kidney, to perform an important sentinel function. However, previous studies of DC function used markers, such as CD11c or CX3CR1, that are not unique to DCs. Here, we evaluated the role of DCs in renal inflammation using a CD11c reporter mouse line and two mouse lines with DC-specific reporters, Zbtb46-GFP and Snx22-GFP. Multiphoton microscopy of kidney sections confirmed that most of the dendritically shaped CD11c+ cells forming a network throughout the renal interstitium expressed macrophage-specific markers. In contrast, DCs marked by Zbtb46-GFP or Snx22-GFP were less abundant, concentrated around blood vessels, and round in shape. We confirmed this pattern of localization using imaging mass cytometry. Motility measurements showed that resident macrophages were sessile, whereas DCs were motile before and after inflammation. Although uninflamed glomeruli rarely contained DCs, injury with nephrotoxic antibodies resulted in accumulation of ZBTB46 + cells in the periglomerular region. ZBTB46 identifies all classic DCs, which can be categorized into two functional subsets that express either CD103 or CD11b. Depletion of ZBTB46 + cells attenuated the antibody-induced kidney injury, whereas deficiency of the CD103+ subset accelerated injury through a mechanism that involved increased neutrophil infiltration. RNA sequencing 7 days after nephrotoxic antibody injection showed that CD11b+ DCs expressed the neutrophil-attracting cytokine CXCL2, whereas CD103+ DCs expressed high levels of several anti-inflammatory genes. These results provide new insights into the distinct functions of the two major DC subsets in glomerular inflammation.

Construction of a viral T2A-peptide based knock-in mouse model for enhanced Cre recombinase activity and fluorescent labeling of podocytes.

Podocyte injury is a key event in glomerular disease leading to proteinuria and opening the path toward glomerular scarring. As a consequence, glomerular research strives to discover molecular mechanisms and signaling pathways affecting podocyte health. The hNphs2.Cre mouse model has been a valuable tool to manipulate podocyte-specific genes and to label podocytes for lineage tracing and purification. Here we designed a novel podocyte-specific tricistronic Cre mouse model combining codon improved Cre expression and fluorescent cell labeling with mTomato under the control of the endogenous Nphs2 promoter using viral T2A-peptides. Independent expression of endogenous podocin, codon improved Cre, and mTomato was confirmed by immunofluorescence, fluorescent activated cell sorting and protein analyses. Nphs2pod.T2A.ciCre.T2A.mTomato/wild-type mice developed normally and did not show any signs of glomerular disease or off-target effects under basal conditions and in states of disease. Nphs2pod.T2A.ciCre.T2A.mTomato/wild-type-mediated gene recombination was superior to conventional hNphs2.Cre mice-mediated gene recombination. Last, we compared Cre efficiency in a disease model by mating Nphs2pod.T2A.ciCre.T2A.mTomato/wild-type and hNphs2.Cre mice to Phb2fl/fl mice. The podocyte-specific Phb2 knockout by Nphs2pod.T2A.ciCre.T2A.mTomato/wild-type mice resulted in an aggravated glomerular injury as compared to a podocyte-specific Phb2 gene deletion triggered by hNphs2.Cre. Thus, we generated the first tricistronic podocyte mouse model combining enhanced Cre recombinase efficiency and fluorescent labeling in podocytes without the need for additional matings with conventional reporter mouse lines.

A role for genetic susceptibility in sporadic focal segmental glomerulosclerosis.

Focal segmental glomerulosclerosis (FSGS) is a syndrome that involves kidney podocyte dysfunction and causes chronic kidney disease. Multiple factors including chemical toxicity, inflammation, and infection underlie FSGS; however, highly penetrant disease genes have been identified in a small fraction of patients with a family history of FSGS. Variants of apolipoprotein L1 (APOL1) have been linked to FSGS in African Americans with HIV or hypertension, supporting the proposal that genetic factors enhance FSGS susceptibility. Here, we used sequencing to investigate whether genetics plays a role in the majority of FSGS cases that are identified as primary or sporadic FSGS and have no known cause. Given the limited number of biopsy-proven cases with ethnically matched controls, we devised an analytic strategy to identify and rank potential candidate genes and used an animal model for validation. Nine candidate FSGS susceptibility genes were identified in our patient cohort, and three were validated using a high-throughput mouse method that we developed. Specifically, we introduced a podocyte-specific, doxycycline-inducible transactivator into a murine embryonic stem cell line with an FSGS-susceptible genetic background that allows shRNA-mediated targeting of candidate genes in the adult kidney. Our analysis supports a broader role for genetic susceptibility of both sporadic and familial cases of FSGS and provides a tool to rapidly evaluate candidate FSGS-associated genes.

Rapid remineralization of multiple disseminated bone lesions after high-dose cytarabine in a patient with isolated myeloid sarcoma.

Isolated myeloid sarcoma is a rare presentation of acute myeloid leukemia. There are limited data available concerning the prognostic relevance and the right treatment strategy for this clinical scenario. Here, we report a case of acute myeloid leukemia with extensive lesions and fractures in multiple bones in a 64-yr-old male patient. Remarkably, treatment with a high-dose cytarabine regimen led to rapid remineralization of all bone lesions and recovery of the patient's mobility within a few weeks. Thereby, surgical treatment and radiotherapy could be avoided, supporting the role of intensive induction and standard consolidation chemotherapy as first-line treatment for myeloid sarcoma.

Intrinsic proinflammatory signaling in podocytes contributes to podocyte damage and prolonged proteinuria.

Inflammation conveys the development of glomerular injury and is a major cause of progressive kidney disease. NF-κB signaling is among the most important regulators of proinflammatory signaling. Its role in podocytes, the epithelial cells at the kidney filtration barrier, is poorly understood. Here, we inhibited NF-κB signaling in podocytes by specific ablation of the NF-κB essential modulator (NEMO, IKKγ). Podocyte-specific NEMO-deficient mice (NEMO(pko)) were viable and did not show proteinuria or overt changes in kidney morphology. After induction of glomerulonephritis, both NEMO(pko) and control mice developed significant proteinuria. However, NEMO(pko) mice recovered much faster, showing rapid remission of proteinuria and restoration of podocyte morphology. Interestingly, quantification of infiltrating macrophages, T-lymphocytes, and granulocytes at day 7 revealed no significant difference between wild-type and NEMO(pko). To further investigate the underlying mechanisms, we created a stable NEMO knockdown mouse podocyte cell line. Again, no overt changes in morphology were observed. Translocation of NF-κB to the nucleus after stimulation with TNFα or IL-1 was sufficiently inhibited. Moreover, secretion of proinflammatory chemokines from podocytes after stimulation with TNFα or IL-1 was significantly reduced in NEMO-deficient podocytes and in glomerular samples obtained at day 7 after induction of nephrotoxic nephritis. Collectively, these results show that proinflammatory activity of NF-κB in podocytes aggravates proteinuria in experimental glomerulonephritis in mice. Based on these data, it may be speculated that immunosuppressive drugs may not only target professional immune cells but also podocytes directly to convey their beneficial effects in various types of glomerulonephritis.