Management of Immune Thrombotic Thrombocytopenic Purpura without Therapeutic Plasma Exchange.

Immune thrombotic thrombocytopenic purpura (iTTP) is a rare, life-threatening autoimmune disorder caused by ADAMTS13 deficiency. Caplacizumab, an anti-VWF nanobody, is approved for iTTP treatment, reducing the need for therapeutic plasma exchange (TPE) and improving platelet count recovery and survival. We conducted a retrospective study on 42 acute iTTP cases in Austria and Germany, treated with a modified regimen aimed at avoiding TPE if platelet count increased after the first caplacizumab dose. Baseline characteristics and patient outcomes were compared with a control group of 59 patients with iTTP, receiving frontline treatment with TPE, caplacizumab, and immunosuppression. The main outcome was the time to platelet count normalization. Secondary outcomes included clinical response, exacerbation, refractory iTTP, iTTP-related deaths, and the time to platelet count doubling. The median time to platelet count normalization was similar between the two cohorts (3 and 4 days; P = 0.31). There were no significant differences in clinical response, exacerbations, refractoriness, iTTP-related deaths, or time to platelet count doubling reflecting the short-term treatment response. Four patients did not respond to the first caplacizumab dose and TPE was subsequently initiated. Cytomegalovirus infection, HIV/hepatitis B co-infection, an ovarian teratoma with associated anti-platelet antibodies, and multiple platelet transfusion before the correct diagnosis may have impeded immediate treatment response in these patients. In conclusion, caplacizumab and immunosuppression alone, without TPE, rapidly controlled thrombotic microangiopathy and achieved a sustained clinical response in iTTP. Our study provides a basis for TPE-free iTTP management in experienced centers via shared decision-making between patients and treating physicians.

Correlation between kidney function and mortality in pyogenic spondylodiscitis: the glomerular filtration rate (GFR) as new predictive parameter?

OBJECTIVE: Pyogenic spondylodiscitis is a severe medical condition, often requiring surgical intervention. Numerous risk factors are known, such as obesity, neurological impairment and old age. In-hospital mortality remains high, therefore other factors may be contributing to the increased mortality. To evaluate kidney function as a risk factor for increased morbidity of pyogenic spondylodiscitis, the glomerular filtration rate (GFR) was correlated with the patients' clinical course. MATERIALS AND METHODS: We retrospectively reviewed the cases of 366 patients and 255 were included for analysis. Clinical, laboratory and surgical data were recorded with a minimum follow-up of three months. For clinical outcome measurement, mortality, length of stay and perioperative complications were analysed. RESULTS: The study included 255 patients (173 men, 82 women; mean age 66.3 years). Patients with a GFR < 59 mL/min spent an average of 5 days longer in the hospital than those with a GFR ≥ 60 mL/min (p = 0.071). The mortality rate increased significantly with a decrease in GFR: A GFR of 30-59 mL/min had a mortality rate of 17.6%, whereas a GFR of < 29 mL/min had one of 30.4% (p = 0.003). Patients with impaired GFR showed an increased rate of postoperative complications (OR 4.7 p = 0.002) and higher rate of intensive care unit (ICU) stay (OR 8.7 p = < 0.001). DISCUSSION: Preoperative GFR values showed a significant correlation with in-hospital mortality in patients with spondylodiscitis, when graded according to the KDIGO stages. Furthermore, a GFR of < 29 ml/mL contributes to a longer ICU stay, postoperative complications and a longer total hospital stay. Therefore, the preoperative GFR could be a marker of kidney function and as a valuable predictive risk factor regarding the clinical in-hospital course of patients suffering from pyogenic spondylodiscitis.

Scaffold polarity proteins Par3A and Par3B share redundant functions while Par3B acts independent of atypical protein kinase C/Par6 in podocytes to maintain the kidney filtration barrier.

Glomerular diseases are a major cause for chronic kidney disorders. In most cases podocyte injury is causative for disease development. Cytoskeletal rearrangements and morphological changes are hallmark features of podocyte injury and result in dedifferentiation and loss of podocytes. Here, we establish a link between the Par3 polarity complex and actin regulators necessary to establish and maintain podocyte architecture by utilizing mouse and Drosophila models to characterize the functional role of Par3A and Par3B and its fly homologue Bazooka in vivo. Only simultaneous inactivation of both Par3 proteins caused a severe disease phenotype. Rescue experiments in Drosophila nephrocytes revealed atypical protein kinase C (aPKC)-Par6 dependent and independent effects. While Par3A primarily acts via aPKC-Par6, Par3B function was independent of Par6. Actin-associated synaptopodin protein levels were found to be significantly upregulated upon loss of Par3A/B in mouse podocytes. Tropomyosin2, which shares functional similarities with synaptopodin, was also elevated in Bazooka depleted nephrocytes. The simultaneous depletion of Bazooka and Tropomyosin2 resulted in a partial rescue of the Bazooka knockdown phenotype and prevented increased Rho1-GTP, a member of a GTPase protein family regulating the cytoskeleton. The latter contribute to the nephrocyte phenotype observed upon loss of Bazooka. Thus, we demonstrate that Par3 proteins share a high functional redundancy but also have specific functions. Par3A acts in an aPKC-Par6 dependent way and regulates RhoA-GTP levels, while Par3B exploits Par6 independent functions influencing synaptopodin localization. Hence, Par3A and Par3B link elements of polarity signaling and actin regulators to maintain podocyte architecture.

European Renal Best Practice endorsement of guidelines for diagnosis and therapy of thrombotic thrombocytopaenic purpura published by the International Society on Thrombosis and Haemostasis.

Thrombotic thrombocytopenic purpura (TTP) is a life-threatening disease that is caused by severe ADAMTS-13 deficiency. Immune-mediated TTP develops due to autoantibodies against ADAMTS-13, whereas congenital TTP is caused by mutations in the ADAMTS13 gene. Diagnostic possibilities and treatment options in TTP have emerged in recent years, which prompted the International Society on Thrombosis and Haemostasis (ISTH) to publish clinical practice guidelines for the diagnosis and treatment of TTP in 2020. In this article, the European Renal Best Practice Working Group endorsed the ISTH guidelines and emphasizes a number of considerations, including the importance of rapid ADAMTS-13 activity testing, the use of rituximab and anti-von Willebrand factor therapies such as caplacizumab, that enhance the clinical applicability of the guidelines in Europe.

Right Heart Function in Cardiorenal Syndrome.

PURPOSE OF REVIEW: Since CRS is critically dependent on right heart function and involved in interorgan crosstalk, assessment and monitoring of both right heart and kidney function are of utmost importance for clinical outcomes. This systematic review aims to comprehensively report on novel diagnostic and therapeutic paradigms that are gaining importance for the clinical management of the growing heart failure population suffering from CRS. RECENT FINDINGS: Cardiorenal syndrome (CRS) in patients with heart failure is associated with poor outcome. Although systemic venous congestion and elevated central venous pressure have been recognized as main contributors to CRS, they are often neglected in clinical practice. The delicate hemodynamic balance in CRS is particularly determined by the respective status of the right heart. The consideration of hemodynamic and CRS profiles is advantageous in tailoring treatment for better preservation of renal function. Assessment and monitoring of right heart and renal function by known and emerging tools like renal Doppler ultrasonography or new biomarkers may have direct clinical implications.

Proteome Analysis of Isolated Podocytes Reveals Stress Responses in Glomerular Sclerosis.

BACKGROUND: Understanding podocyte-specific responses to injury at a systems level is difficult because injury leads to podocyte loss or an increase of extracellular matrix, altering glomerular cellular composition. Finding a window into early podocyte injury might help identify molecular pathways involved in the podocyte stress response. METHODS: We developed an approach to apply proteome analysis to very small samples of purified podocyte fractions. To examine podocytes in early disease states in FSGS mouse models, we used podocyte fractions isolated from individual mice after chemical induction of glomerular disease (with Doxorubicin or LPS). We also applied single-glomerular proteome analysis to tissue from patients with FSGS. RESULTS: Transcriptome and proteome analysis of glomeruli from patients with FSGS revealed an underrepresentation of podocyte-specific genes and proteins in late-stage disease. Proteome analysis of purified podocyte fractions from FSGS mouse models showed an early stress response that includes perturbations of metabolic, mechanical, and proteostasis proteins. Additional analysis revealed a high correlation between the amount of proteinuria and expression levels of the mechanosensor protein Filamin-B. Increased expression of Filamin-B in podocytes in biopsy samples from patients with FSGS, in single glomeruli from proteinuric rats, and in podocytes undergoing mechanical stress suggests that this protein has a role in detrimental stress responses. In Drosophila, nephrocytes with reduced filamin homolog Cher displayed altered filtration capacity, but exhibited no change in slit diaphragm structure. CONCLUSIONS: We identified conserved mechanisms of the podocyte stress response through ultrasensitive proteome analysis of human glomerular FSGS tissue and purified native mouse podocytes during early disease stages. This approach enables systematic comparisons of large-scale proteomics data and phenotype-to-protein correlation.

Protein half-life determines expression of proteostatic networks in podocyte differentiation.

Podocytes are highly specialized, epithelial, postmitotic cells, which maintain the renal filtration barrier. When adapting to considerable metabolic and mechanical stress, podocytes need to accurately maintain their proteome. Immortalized podocyte cell lines are a widely used model for studying podocyte biology in health and disease in vitro. In this study, we performed a comprehensive proteomic analysis of the cultured human podocyte proteome in both proliferative and differentiated conditions at a depth of >7000 proteins. Similar to mouse podocytes, human podocyte differentiation involved a shift in proteostasis: undifferentiated podocytes have high expression of proteasomal proteins, whereas differentiated podocytes have high expression of lysosomal proteins. Additional analyses with pulsed stable-isotope labeling by amino acids in cell culture and protein degradation assays determined protein dynamics and half-lives. These studies unraveled a globally increased stability of proteins in differentiated podocytes. Mitochondrial, cytoskeletal, and membrane proteins were stabilized, particularly in differentiated podocytes. Importantly, protein half-lives strongly contributed to protein abundance in each state. These data suggest that regulation of protein turnover of particular cellular functions determines podocyte differentiation, a paradigm involving mitophagy and, potentially, of importance in conditions of increased podocyte stress and damage.-Schroeter, C. B., Koehler, S., Kann, M., Schermer, B., Benzing, T., Brinkkoetter, P. T., Rinschen, M. M. Protein half-life determines expression of proteostatic networks in podocyte differentiation.

Proline-dependent and basophilic kinases phosphorylate human TRPC6 at serine 14 to control channel activity through increased membrane expression.

Signaling via the transient receptor potential (TRP) ion channel C6 plays a pivotal role in hereditary and sporadic glomerular kidney disease. Several studies have identified gain-of-function mutations of TRPC6 and report induced expression and enhanced channel activity of TRPC6 in association with glomerular diseases. Interfering with TRPC6 activity may open novel therapeutic pathways. TRPC6 channel activity is controlled by protein expression and stability as well as intracellular trafficking. Identification of regulatory phosphorylation sites in TRPC6 and corresponding protein kinases is essential to understand the regulation of TRPC6 activity and may result in future therapeutic strategies. In this study, an unbiased phosphoproteomic screen of human TRPC6 identified several novel serine phosphorylation sites. The phosphorylation site at serine 14 of TRPC6 is embedded in a basophilic kinase motif that is highly conserved across species. We confirmed serine 14 as a target of MAPKs and proline-directed kinases like cyclin-dependent kinase 5 (Cdk5) in cell-based as well as in vitro kinase assays and quantitative phosphoproteomic analysis of TRPC6. Phosphorylation of TRPC6 at serine 14 enhances channel conductance by boosting membrane expression of TRPC6, whereas protein stability and multimerization of TRPC6 are not altered, making serine 14 phosphorylation a potential drug target to interfere with TRPC6 channel activity.-Hagmann, H., Mangold, N., Rinschen, M. M., Koenig, T., Kunzelmann, K., Schermer, B., Benzing, T., Brinkkoetter, P. T. Proline-dependent and basophilic kinases phosphorylate human TRPC6 at serine 14 to control channel activity through increased membrane expression.

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.

Prohibitin-2 Depletion Unravels Extra-Mitochondrial Functions at the Kidney Filtration Barrier.

Mitochondrial fusion is essential for maintenance of mitochondrial function and requires the prohibitin ring complex subunit prohibitin-2 (PHB2) at the mitochondrial inner membrane. Loss of the stomatin/PHB/flotillin/HflK/C (SPFH) domain containing protein PHB2 causes mitochondrial dysfunction and defective mitochondria-mediated signaling, which is implicated in a variety of human diseases, including progressive renal disease. Here, we provide evidence of additional, extra-mitochondrial functions of this membrane-anchored protein. Immunofluorescence and immunogold labeling detected PHB2 at mitochondrial membranes and at the slit diaphragm, a specialized cell junction at the filtration slit of glomerular podocytes. PHB2 coprecipitated with podocin, another SPFH domain-containing protein, essential for the assembly of the slit diaphragm protein-lipid supercomplex. Consistent with an evolutionarily conserved extra-mitochondrial function, the ortholog of PHB2 in Caenorhabditis elegans was also not restricted to mitochondria but colocalized with the mechanosensory complex that requires the podocin ortholog MEC2 for assembly. Knockdown of phb-2 partially phenocopied loss of mec-2 in touch neurons of the nematode, resulting in impaired gentle touch sensitivity. Collectively, these data indicate that, besides its established role in mitochondria, PHB2 may have an additional function in conserved protein-lipid complexes at the plasma membrane.

Metabolism and homeostasis in the kidney: metabolic regulation through insulin signaling in the kidney.

Metabolic signaling pathways orchestrate the dynamic turnover between catabolic and anabolic processes. Thereby, they ensure the viability of the cell and assure proper function of the tissue in changing environments regarding the availability of nutrients. Yet, renal cells are not considered to be prime targets of metabolic signaling. Research of the last decade has proposed new roles of specifically altered metabolic signaling pathways. In particular, the insulin signaling cascade, a potent regulator of cellular metabolism and energy homeostasis, seems to be implicated in the progression of diabetic and non-diabetic kidney disease. The aim of this review is to summarize the current knowledge on metabolic signaling events in different renal compartments in states of health and disease. We will focus on the role of insulin signaling events and highlight recent advances in the understanding of the regulatory interplay between insulin signaling and mitochondrial function contributing to the pathogenesis of kidney disease.

Prohibitin Signaling at the Kidney Filtration Barrier.

The kidney filtration barrier consists of three well-defined anatomic layers comprising a fenestrated endothelium, the glomerular basement membrane (GBM) and glomerular epithelial cells, the podocytes. Podocytes are post-mitotic and terminally differentiated cells with primary and secondary processes. The latter are connected by a unique cell-cell contact, the slit diaphragm. Podocytes maintain the GBM and seal the kidney filtration barrier to prevent the onset of proteinuria. Loss of prohibitin-1/2 (PHB1/2) in podocytes results not only in a disturbed mitochondrial structure but also in an increased insulin/IGF-1 signaling leading to mTOR activation and a detrimental metabolic switch. As a consequence, PHB-knockout podocytes develop proteinuria and glomerulosclerosis and eventually loss of renal function. In addition, experimental evidence suggests that PHB1/2 confer additional, extra-mitochondrial functions in podocytes as they localize to the slit diaphragm and thereby stabilize the unique intercellular contact between podocytes required to maintain an effective filtration barrier.

Quantitative deep mapping of the cultured podocyte proteome uncovers shifts in proteostatic mechanisms during differentiation.

The renal filtration barrier is maintained by the renal podocyte, an epithelial postmitotic cell. Immortalized mouse podocyte cell lines-both in the differentiated and undifferentiated state-are widely utilized tools to estimate podocyte injury and cytoskeletal rearrangement processes in vitro. Here, we mapped the cultured podocyte proteome at a depth of more than 8,800 proteins and quantified 7,240 proteins. Copy numbers of proteins mutated in forms of hereditary nephrotic syndrome or focal segmental glomerulosclerosis (FSGS) were assessed. We found that cultured podocytes express abundant copy numbers of endogenous receptors, such as tyrosine kinase membrane receptors, the G protein-coupled receptor (GPCR), NPR3 (ANP receptor), and several poorly characterized GPCRs. The data set was correlated with deep mapping mRNA sequencing ("mRNAseq") data from the native mouse podocyte, the native mouse podocyte proteome and staining intensities from the human protein atlas. The generated data set was similar to these previously published resources, but several native and high-abundant podocyte-specific proteins were not identified in the data set. Notably, this data set detected general perturbations in proteostatic mechanisms as a dominant alteration during podocyte differentiation, with high proteasome activity in the undifferentiated state and markedly increased expression of lysosomal proteins in the differentiated state. Phosphoproteomics analysis of mouse podocytes at a resolution of more than 3,000 sites suggested a preference of phosphorylation of actin filament-associated proteins in the differentiated state. The data set obtained here provides a resource and provides the means for deep mapping of the native podocyte proteome and phosphoproteome in a similar manner.

Par3A is dispensable for the function of the glomerular filtration barrier of the kidney.

Polarity signaling through the atypical PKC (aPKC)-Par polarity complex is essential for the development and maintenance of the podocyte architecture and the function of the glomerular filtration barrier of the kidney. To study the contribution of Par3A in this complex, we generated a novel Pard3 podocyte-specific knockout mouse model by targeting exon 6 of the Pard3 gene. Genetic deletion of Pard3a did not impair renal function, neither at birth nor later in life. Even challenging the animals did not result in glomerular disease. Despite its well-established role in aPKC-mediated signaling, Par3A appears to be dispensable for the function of the glomerular filtration barrier. Moreover, its homolog Pard3b, and not Pard3a, is the dominant Par3 gene expressed in podocytes and found at the basis of the slit diaphragm, where it partially colocalizes with podocin. In conclusion, Par3A function is either dispensable for slit diaphragm integrity, or compensatory mechanisms and a high redundancy of the different polarity proteins, including Par3B, Lgl, or PALS1, maintain the function of the glomerular filtration barrier, even in the absence of Par3A.

Comparative phosphoproteomic analysis of mammalian glomeruli reveals conserved podocin C-terminal phosphorylation as a determinant of slit diaphragm complex architecture.

Glomerular biology is dependent on tightly controlled signal transduction networks that control phosphorylation of signaling proteins such as cytoskeletal regulators or slit diaphragm proteins of kidney podocytes. Cross-species comparison of phosphorylation events is a powerful mean to functionally prioritize and identify physiologically meaningful phosphorylation sites. Here, we present the result of phosphoproteomic analyses of cow and rat glomeruli to allow cross-species comparisons. We discovered several phosphorylation sites with potentially high biological relevance, e.g. tyrosine phosphorylation of the cytoskeletal regulator synaptopodin and the slit diaphragm protein neph-1 (Kirrel). Moreover, cross-species comparisons revealed conserved phosphorylation of the slit diaphragm protein nephrin on an acidic cluster at the intracellular terminus and conserved podocin phosphorylation on the very carboxyl terminus of the protein. We studied a highly conserved podocin phosphorylation site in greater detail and show that phosphorylation regulates affinity of the interaction with nephrin and CD2AP. Taken together, these results suggest that species comparisons of phosphoproteomic data may reveal regulatory principles in glomerular biology. All MS data have been deposited in the ProteomeXchange with identifier PXD001005 (http://proteomecentral.proteomexchange.org/dataset/PXD001005).

Cyclin I and p35 determine the subcellular distribution of Cdk5.

The atypical cyclin-dependent kinase 5 (Cdk5) serves an array of different functions in cell biology. Among these are axonal guidance, regulation of intercellular contacts, cell differentiation, and prosurvival signaling. The variance of these functions suggests that Cdk5 activation comes to pass in different cellular compartments. The kinase activity, half-life, and substrate specificity of Cdk5 largely depend on specific activators, such as p25, p35, p39, and cyclin I. We hypothesized that the subcellular distribution of Cdk5 activators also determines the localization of the Cdk5 protein and sets the stage for targeted kinase activity within distinct cellular compartments to suit the varying roles of Cdk5. Cdk5 localization was analyzed in murine kidney and brain slices of wild-type and cyclin I- and/or p35-null mice by immunohistochemistry and in cultured mouse podocytes using immunofluorescence labeling, as well as cell fractionation experiments. The predominance of cyclin I mediates the nuclear localization of Cdk5, whereas the predominance of p35 results in a membranous localization of Cdk5. These findings were further substantiated by overexpression of cyclin I and p35 with altered targeting characteristics in human embryonic kidney 293T cells. These studies reveal that the subcellular localization of Cdk5 is determined by its specific activators. This results in the directed Cdk5 kinase activity in specific cellular compartments dependent on the activator present and allows Cdk5 to serve multiple independent roles.

The NF-κB essential modulator (NEMO) controls podocyte cytoskeletal dynamics independently of NF-κB.

Maintenance of the glomerular filtration barrier with its fenestrated endothelium, the glomerular basement membrane, and the podocytes as the outer layer, is a major prerequisite for proper renal function. Tight regulation of the balance between plasticity and rigidity of the podocytes' architecture is required to prevent the onset of glomerular disease, mainly proteinuria. The underlying cellular signaling pathways that regulate the organization of the podocytes' cytoskeleton are still a matter of controversial debate. In this study, we investigated the role of the NF-κB signaling pathway in podocyte cytoskeletal dynamics. As previously published, genetic inhibition of the NF-κB essential modulator (NEMO) in podocytes does not affect glomerular function under physiological, nonstressed conditions nor does it alter the initial podocyte response in an experimental glomerulonephritis (NTN) model (Brähler S, Ising C, Hagmann H, Rasmus M, Hoehne M, Kurschat C, Kisner T, Goebel H, Shankland SJ, Addicks K, Thaiss F, Schermer B, Pasparakis M, Benzing T, Brinkkoetter PT. Am J Physiol Renal Physiol 303: F1473-F1475, 2012). Quite the contrary, podocyte-specific NEMO null mice recovered significantly faster and did not develop glomerulosclerosis and end-stage renal failure over time. Here, we show that cytoskeletal rearrangements and increased podocyte motility following stimulation with IL-1, TNF-α, or LPS depend on NEMO. NEMO also regulates the phosphorylation of the MAP kinase ERK1/2 and suppresses the activation of RhoA following stimulation with IL-1. The migratory response and altered ERK1/2 phosphorylation is independent of NF-κB signaling as demonstrated by expression of a mutant IκB resistant to phosphorylation and degradation. In conclusion, signaling through NEMO might not only be involved in the production of NF-κB proinflammatory chemokines but also regulates podocyte dynamics independently of NF-κB, most likely through small GTPases and MAP kinases.