P17-A140†Clinical outcomes of the first organ-cultured preloaded DMEK-a single centre studie.

The use of ready-to-use grafts from specialized eye banks might provide a number of benefits, including graft quality control, assured availability at a certain operation time, a decreased likelihood of case cancellation, and a shorter and less sophisticated DMEK surgery, with a resulting lower risk of graft damage. However, it is critical to thoroughly establish the clinical safety of employing these preloaded tissues. Especially since most of the studies were prepared and stored under hypothermic conditions. There are only a few studies on preprepared tissues in organ culture, which are partly controversial.In this prospective study we included patients who received DMEK surgery at the Knappschaft Eye Clinic Sulzbach. Patients received either a preloaded DMEK (pDMEK), prepared five days before surgery in the eye bank, or a conventional, directly before surgery, surgeon-prepared DMEK (sDMEK).The preliminary data show a trend towards more frequent need for rebubbing in the pDMEK group and a statistically non-significant lower postoperative endothelial cell count compared to the sDMEK group. However, the development of visual acuity and decrease in corneal thickness is comparable in both groups.Therefore, we investigated the clinical outcomes of the first organ-cultured preloaded DMEK cases and compared these outcomes with those from our very last cases with surgically loaded tissues from a single centre.

Comparison of clinical outcomes after precut DMEK with or without dextran-containing medium compared to standard DMEK: a prospective pilot study.

PURPOSE: To investigate the clinical outcome and complication rate of precut Descemet membrane endothelial keratoplasty (DMEK) in two different culture conditions, dextran-containing and dextran-free medium, and compare the results with the current standard DMEK procedure. METHODS: A prospective study of 32 eyes suffering from Fuchs endothelial dystrophy were scheduled for DMEK with a follow-up of one year. The eyes were divided into four subgroups. Group + D (n = 7) received a precut DMEK stored in dextran-containing transport medium, and Group - D (n = 9) received a precut DMEK without dextran-containing medium. The respective fellow eyes received a standard DMEK (S) (preparation directly prior to surgery) stored in dextran-containing medium (S-D + ; n = 7) or without (S-D-; n = 9). RESULTS: Clinical outcome (visual acuity, endothelial cell count, central corneal thickness) and rebubbling rate were comparable for all four groups. None of the patients had a graft failure. CONCLUSION: The preliminary data of the pilot study show that precut liquid-bubble DMEK leads to comparable clinical results regardless of dextran-containing or dextran-free organ culture medium and is further comparable to the standard DMEK procedure.

Cleavage plane after liquid-bubble preparation of Descemet's membrane.

PURPOSE: To investigate the ultrastructure of the cleavage plane of human cornea after liquid-bubble-prepared tissue for Descemet's membrane endothelial keratoplasty (DMEK). METHODS: Experimental study with scanning electron microscopy (SEM) and block-face SEM of 18 corneal specimens. Fresh human research donor corneoscleral discs (n = 12) were prepared with liquid-bubble technique or examined as untreated controls (n = 3). In addition, Descemet's membrane samples, n = 3, were obtained in DMEK surgery. RESULTS: The cleavage plane after liquid-bubble Descemet's membrane (DM) preparation was consistently located between interfacial matrix and posterior stromal collagen lamellae, providing a largely smooth surface exposing the amorphous interfacial zone without any significant amounts of adherent stromal remnants. No demarcation of a distinct pre-DM layer could be detected. CONCLUSION: The DMEK graft preparation performed by liquid-bubble technique showed a smooth cleavage plane and could not reveal any demarcation of a distinct pre-DM layer.

Comparison of preloaded grafts for Descemet membrane endothelial keratoplasty (DMEK) in a novel preloaded transport cartridge compared to conventional precut grafts.

To determine the safety and graft quality of eye bank precut and preloaded grafts for Descemet membrane endothelial keratoplasty (DMEK) after storage and shipping in a novel preloaded transport cartridge compared to precut grafts in a conventional viewing chamber. In this laboratory proof-of-concept study, 29 human donor corneas that were unsuitable for transplantation with a mean endothelial cell density of 1948 ± 260 cells/mm2 were prepared using liquid bubble technique for producing precut lamellar grafts. The grafts were either preloaded into novel transport cartridge (n = 16) or transferred into conventional Krolman viewing chamber (control, n = 13). Grafts were stored for 24 or 48 h in dextran-containing medium at room temperature and subjected to a shipping simulation. Endothelial cell loss (ECL) and morphology were determined at different steps. Endothelial cell viability staining was performed with calcein dye. Mean ECL in the preloaded transport cartridge was 0.7% ± 1.2% after 24 h and 3.4% ± 1.2% (p = 0.006) after 48 h storage and injection. In the control group the ECL was mean 1.6% ± 2.7% after 24 h compared to 3.7% ± 0.9% (p = 0.042) after 48 h. The slightly higher endothelial cell loss in the viewing chamber group after 48 h was not statistically significant compared to the preloaded transport cartridge (p = 0.8). Calcein staining was comparably low in all groups and correlated with the low ECL in both groups. DMEK grafts can be preloaded into a novel transport cartridge using a "no touch" technique, stored and shipped for up to 2 days in dextran-containing medium without significant ECL.

Alginate- and Hyaluronic Acid-Based Hydrogels as Vitreous Substitutes: An In Vitro Evaluation.

Purpose: To study alginate- and hyaluronic acid-based hydrogels in vitro as vitreous substitutes. Methods: Biopolymeric hydrogels based on high-molecular alginate (0.5% and 1.0%) and hyaluronic acid (1.0% and Healaflow) were compared with extracted human vitreous bodies and silicone oil (SIL-5000) regarding their optical properties (refractive index, transmission) and viscoelastic characteristics (storage modulus G', loss modulus G″). The cytotoxic (metabolic activity, apoptosis) and antiproliferative profiles were determined using cultured human fibroblasts, ARPE-19, and photoreceptor cells. The hydrogel systems were applied to human fetal retinal pigment epithelial cells cultured for two months until maximum transepithelial electrical resistance (TEER) to investigate the effect of the gel matrices on tight junctions using TEER measurements and immunostainings against the tight junction protein ZO-1. Results: Tested alginate- and hyaluronic acid-based hydrogels resembled the natural refractive index of human vitreous bodies (1.3356-1.3360) in contrast to SIL-5000 (1.4034) and showed high optical transparency (>90%) within the visible light region. The biopolymeric hydrogels exhibited viscoelastic properties similar to juvenile vitreous bodies with G'>G″ adjustable via different gelation times, contrary to SIL-5000 (G'

Analysis of different types of anesthesia in descemet membrane endothelial keratoplasty.

PURPOSE: The purpose of this study is to compare the safety and clinical results of descemet membrane endothelial keratoplasty (DMEK) in topical, peribulbar or general anesthesia. METHODS: This is a retrospective, post hoc matched study of 120 patients having received DMEK surgery with different types of anesthesia (n = 40 topical, n = 40 peribulbar, n = 40 general anesthesia). Endpoint criteria were intraoperative complications, endothelial cell count, central corneal thickness and graft rejection rate, rebubbling rate and best-corrected visual acuity after 1, 3 and 6 months. RESULTS: The group with topical anesthesia showed more often intraoperative difficulties such as vitreous pressure (p < 0.05), difficult graft unfolding (p = 0.14) and patient restlessness (p = 0.07). However, all three groups achieved comparable functional visual results after 6 months (p = 0.96). CONCLUSION: DMEK in topical anesthesia is feasible and shows comparable final visual results but should be restricted to selected cooperative patients and performed by experienced surgeons due to possible higher intraoperative challenges.

Age-Related Loss of Human Vitreal Viscoelasticity.

PURPOSE: To determine the viscoelasticity of human vitreous bodies and its changes with age in order to benefit the understanding and therapy of vitreoretinal diseases. METHODS: In a postmortem study, 190 human vitreous bodies were extracted from 33- to 92-year-old donors, analyzed with regard to their viscoelastic properties via dynamic mechanical analyses, and compared with bovine and porcine vitreous. Postmortem intervals and donor-related parameters were examined as potential parameters influencing vitreous viscoelasticity. Dynamic moduli of different hyaluronic acid (HA) solutions as well as human vitreous treated with HA injections were determined by frequency sweep tests. RESULTS: With age the viscoelasticity of human vitreous bodies decreased significantly and independently of postmortem intervals, diabetes, and the donor's sex. The storage modulus G' and loss modulus G″ correlated strongly with the donor's age with r = -0.789 and r = -0.764, respectively. Bovine and porcine vitreous bodies exhibited dynamic moduli comparable only to the viscoelastic properties of aged human vitreous and are thus limited models for the simulation of the human vitreous. The viscoelasticity of aged human vitreous bodies was found to be increased after intravitreal injections of highly concentrated HA. CONCLUSIONS: The present postmortem study is the first to show a significant age-related reduction in the viscoelasticity of entire human vitreous bodies. Highly concentrated HA injections may serve as a possible therapeutic approach for restoring the viscoelasticity of aged vitreous bodies. TRANSLATIONAL RELEVANCE: These findings improve the understanding and therapy of the vitreous liquefaction with age and the associated vitreoretinal diseases.

Significant differences between specular microscopy and corneal bank endothelial cell counts - a pilot study.

BACKGROUND: It was shown recently that endothelial cell count performed by cornea banks overestimates the real number of endothelial cells. The aim of this study was to investigate the internal quality of preclinical ECD in human donor corneas using two widely used methods for endothelial cell counting, transmitted light microscopy used in organ culture tissue bank and clinically used specular microscopy. METHODS: Twenty human donor corneas that could not be transplanted were included in this analysis. Differences in evaluating endothelial cell density (ECD) and hexagonal endothelial cell ratio (HEX) between clinical specular microscopy (CSM) and corneal bank transmitted light microscope (CBLM) were evaluated as well as differences between automated and manual cell counts. RESULTS: Automated CBLM showed a higher ECD of 31.85% compared to automated CSM, while manual CBLM counting is 10.51% higher compared to manual CSM (p < 0.01). Further, higher average ECD values result in a higher difference between CSM and CBLM measurements. The manual CBLM ECDs were significantly higher compared to automated derived ECD from CSM (p < 0.01). However, no systematic bias can be detected when comparing the differences of the measurements with the average ECD measurements of both methods. CONCLUSION: This preclinical pilot study confirmed a significant higher ECD using transmitted light microscopy in organ culture compared to clinical specular microscopy. This indicates that the early rapid decrease of EC universally observed after surgery might be partly artefactual.

Safety analysis and results of a borosilicate glass cartridge for no-touch graft loading and injection in Descemet membrane endothelial keratoplasty.

PURPOSE: The aim of this study was to investigate the clinical outcome after standardized DMEK using a glass injector. METHODS: A total of 254 patients undergoing DMEK surgery using a disposable DMEK borosilicate glass cartridge system were included in this retrospective study. The mean follow-up time was 13.2 months (SD ± 8.1, range 6-36 months). The used glass cartridge system has an aperture diameter of 1.6 mm and a posterior loading orifice of 4.29 mm. Scanning electron microscopy (SEM) was used for estimation of the surface relief of the glass cartridge and comparison with a standard plastic injector cartridge. RESULTS: Mean endothelial cell count of donor grafts was 2465 cells/mm2 (SD ± 199). After 6 weeks of DMEK endothelial cell count decreased by - 28.6% to 1759 cells/mm2 (SD ± 435) (Wilcoxon p = 0.001) and remained stable at the final follow-up at 1735 cells/mm2 (SD ± 442) (Wilcoxon p = 0.89). SEM showed smoother surface of the glass cartridge in comparison with a plastic cartridge. CONCLUSION: This study showed that this simple and effective DMEK cartridge seems to be a safe and viable device for minimized graft manipulation during DMEK surgery.

Precut DMEK Using Dextran-Containing Storage Medium Is Equivalent to Conventional DMEK: A Prospective Pilot Study.

PURPOSE: To compare the clinical outcome after Descemet membrane endothelial keratoplasty (DMEK) either as precut or conventional Descemet membrane graft preparation under standard European eye bank organ culture conditions. METHODS: This was a prospective pilot study of patients receiving either precut or conventional DMEK. Graft preparation was performed using the liquid bubble technique. Precut grafts (n = 22) were prepared 1 day before surgery in the eye bank and stored in dextran-containing organ culture medium within a transport viewing chamber. Conventional grafts (n = 29) were prepared directly before surgery. End point criteria included the endothelial cell count (ECC), central corneal thickness, graft rejection rate, rebubbling rate, and best-corrected visual acuity after 1, 3, and 6 months. RESULTS: A post hoc matched analysis revealed no statistically significant differences between the 2 groups. The ECC in the precut and conventional groups was comparable with an EC loss of 34% and 35%, respectively, after 6 months. The early graft failure rate, best-corrected visual acuity, and central corneal thickness were comparable between the 2 groups. CONCLUSIONS: This pilot study shows a comparable clinical outcome after DMEK surgery for precut Descemet membrane grafts versus conventionally prepared grafts, using the liquid bubble preparation technique and storage conditions with dextran-containing medium.

Impact of 10% SF6 Gas Compared to 100% Air Tamponade in Descemet's Membrane Endothelial Keratoplasty.

PURPOSE: To compare the clinical outcomes following Descemet's membrane endothelial keratoplasty (DMEK) with 100% air tamponade versus 10% sulfur hexafluoride (SF6) tamponade. METHODS: Retrospective analysis of 108 consecutive DMEK cases subdivided by anterior chamber tamponade with 54 eyes receiving 10% SF6 and 54 eyes receiving 100% air injection. A post-hoc matched analysis revealed no statistically significant differences between the groups. The main outcome measurements were the complication rate, including intra- and postoperative complications and graft detachment rate requiring re-bubbling. Clinical outcome included best-corrected visual acuity (BCVA), endothelial cell count (ECC), and central corneal thickness (CCT) measured 1, 3, and 6 months after DMEK surgery. RESULTS: The graft detachment rate with consecutive re-bubbling was 18.5% in the air group and 22.2% in the SF6 group (p = 0.2). Remaining small peripheral graft detachments with a clear cornea occurred more often in the 100% air group (air: 22.2%; 12/54, 6/12 inferior compared to SF6: 7.4%; 4/54, 2/4 inferior; p = 0.06). The primary graft failure rate was comparable between the two groups. No complete graft detachment occurred. Outcome results for BCVA, ECC, and CCT at all follow-up time points were comparable between the two groups. CONCLUSION: The clinical outcomes (including re-bubbling rate, primary graft failure rate, and endothelial cell loss) were comparable with 100% air versus 10% SF6 tamponade, whereas other studies suggest that a higher SF6 concentration (20%) may result in a lower re-bubbling rate.

Clinical Comparison of Two Methods of Graft Preparation in Descemet Membrane Endothelial Keratoplasty.

PURPOSE: Descemet membrane endothelial keratoplasty (DMEK) has been improved over the last decade. The aim of this study was to compare the clinical outcome of the recently introduced liquid bubble method compared to the standard manual preparation. METHODS: This retrospective study evaluated the outcome of 200 patients after DMEK surgery using two different graft preparation techniques. Ninety-six DMEK were prepared by manual dissection and 104 by the novel liquid bubble technique. The mean follow-up time was 13.7 months (SD ± 8, range 6-36 months). RESULTS: Best corrected mean visual acuity (BCVA) increased for all patients statistically significant from baseline 0.85 logMAR (SD ± 0.5) to 0.26 logMAR (SD ± 0.27) at the final follow-up (Wilcoxon, p = 0.001). Subgroup analyses of BCVA at the final follow-up between manual dissection and liquid bubble preparation showed no statistically significant difference (Mann-Whitney U Test, p = 0.64). The mean central corneal thickness was not statistically different (manual dissection: 539 µm, SD ± 68 µm and liquid bubble technique: 534 µm, SD ± 52 µm,) between the two groups (Mann-Whitney U Test, p = 0.64). At the final follow-up, mean endothelial cell count of donor grafts was statistically not significant different at the final follow-up with 1761 cells/mm2 (-30.7%, SD ± 352) for manual dissection compared to liquid bubble technique with 1749 cells/mm2 (-29.9%, SD ± 501) (Mann-Whitney U-Test, p = 0.73). The re-DMEK rate was comparable for manual dissection with 8 cases (8.3%) and 7 cases (6.7%) for liquid bubble dissection (p = 0.69, Chi-Square Test). CONCLUSION: Regarding the clinical outcome, we did not find a statistical significant difference between manual dissection and liquid bubble graft preparation. Both preparation techniques lead to an equivalent clinical outcome after DMEK surgery.

The Calcineurin-Binding, Activity-Dependent Splice Variant Dynamin1xb Is Highly Enriched in Synapses in Various Regions of the Central Nervous System.

In the present study, we generated and characterized a splice site-specific monoclonal antibody that selectively detects the calcineurin-binding dynamin1 splice variant dynamin1xb. Calcineurin is a Ca2+-regulated phosphatase that enhances dynamin1 activity and is an important Ca2+-sensing mediator of homeostatic synaptic plasticity in neurons. Using this dynamin1xb-specific antibody, we found dynamin1xb highly enriched in synapses of all analyzed brain regions. In photoreceptor ribbon synapses, dynamin1xb was enriched in close vicinity to the synaptic ribbon in a manner indicative of a peri-active zone immunolabeling. Interestingly, in dark-adapted mice we observed an enhanced and selective enrichment of dynamin1xb in both synaptic layers of the retina in comparison to light-adapted mice. This could be due to an illumination-dependent recruitment of dynamin1xb to retinal synapses and/or due to a darkness-induced increase of dynamin1xb biosynthesis. These latter findings indicate that dynamin1xb is part of a versatile and highly adjustable, activity-regulated endocytic synaptic machinery.

Human NACHT, LRR, and PYD domain-containing protein 3 (NLRP3) inflammasome activity is regulated by and potentially targetable through Bruton tyrosine kinase.

BACKGROUND: The Nod-like receptor NACHT, LRR, and PYD domain-containing protein 3 (NLRP3) and Bruton tyrosine kinase (BTK) are protagonists in innate and adaptive immunity, respectively. NLRP3 senses exogenous and endogenous insults, leading to inflammasome activation, which occurs spontaneously in patients with Muckle-Wells syndrome; BTK mutations cause the genetic immunodeficiency X-linked agammaglobulinemia (XLA). However, to date, few proteins that regulate NLRP3 inflammasome activity in human primary immune cells have been identified, and clinically promising pharmacologic targeting strategies remain elusive. OBJECTIVE: We sought to identify novel regulators of the NLRP3 inflammasome in human cells with a view to exploring interference with inflammasome activity at the level of such regulators. METHODS: After proteome-wide phosphoproteomics, the identified novel regulator BTK was studied in human and murine cells by using pharmacologic and genetic BTK ablation. RESULTS: Here we show that BTK is a critical regulator of NLRP3 inflammasome activation: pharmacologic (using the US Food and Drug Administration-approved inhibitor ibrutinib) and genetic (in patients with XLA and Btk knockout mice) BTK ablation in primary immune cells led to reduced IL-1ß processing and secretion in response to nigericin and the Staphylococcus aureus toxin leukocidin AB (LukAB). BTK affected apoptosis-associated speck-like protein containing a CARD (ASC) speck formation and caspase-1 cleavage and interacted with NLRP3 and ASC. S aureus infection control in vivo and IL-1ß release from cells of patients with Muckle-Wells syndrome were impaired by ibrutinib. Notably, IL-1ß processing and release from immune cells isolated from patients with cancer receiving ibrutinib therapy were reduced. CONCLUSION: Our data suggest that XLA might result in part from genetic inflammasome deficiency and that NLRP3 inflammasome-linked inflammation could potentially be targeted pharmacologically through BTK.

The Disease Protein Tulp1 Is Essential for Periactive Zone Endocytosis in Photoreceptor Ribbon Synapses.

Mutations in the Tulp1 gene cause severe, early-onset retinitis pigmentosa (RP14) in humans. In the retina, Tulp1 is mainly expressed in photoreceptors that use ribbon synapses to communicate with the inner retina. In the present study, we demonstrate that Tulp1 is highly enriched in the periactive zone of photoreceptor presynaptic terminals where Tulp1 colocalizes with major endocytic proteins close to the synaptic ribbon. Analyses of Tulp1 knock-out mice demonstrate that Tulp1 is essential to keep endocytic proteins enriched at the periactive zone and to maintain high levels of endocytic activity close to the synaptic ribbon. Moreover, we have discovered a novel interaction between Tulp1 and the synaptic ribbon protein RIBEYE, which is important to maintain synaptic ribbon integrity. The current findings suggest a new model for Tulp1-mediated localization of the endocytic machinery at the periactive zone of ribbon synapses and offer a new rationale and mechanism for vision loss associated with genetic defects in Tulp1.

ArfGAP3 is a component of the photoreceptor synaptic ribbon complex and forms an NAD(H)-regulated, redox-sensitive complex with RIBEYE that is important for endocytosis.

Ribbon synapses are tonically active synapses in the retina and inner ear with intense vesicle traffic. How this traffic is organized and regulated is still unknown. Synaptic ribbons, large presynaptic structures associated with numerous synaptic vesicles, appear to be essential for this process. The base of the synaptic ribbon is anchored at the active zone and is a hotspot of exocytosis. The synaptic ribbon complex is also important for vesicle replenishment. RIBEYE is a unique and major component of synaptic ribbons. It consists of a unique A-domain and an NAD(H)-binding, C-terminal B-domain. In the present study, we show that the Arf-GTPase activating protein-3 (ArfGAP3), a well characterized regulator of vesicle formation at the Golgi apparatus, is also a component of the synaptic ribbon complex in photoreceptor synapses of the mouse retina and interacts with RIBEYE as shown by multiple, independent approaches. ArfGAP3 binds to RIBEYE(B)-domain in an NAD(H)-dependent manner. The interaction is redox sensitive because NADH is more efficient than the oxidized NAD(+) in promoting ArfGAP3-RIBEYE interaction. RIBEYE competes with the GTP-binding protein Arf1 for binding to ArfGAP3. Thus, binding of RIBEYE(B) to ArfGAP3 could prevent inactivation of Arf1 by ArfGAP3 and provides the synaptic ribbon with the possibility to control Arf1 function. The interaction is relevant for endocytic vesicle trafficking because overexpression of ArfGAP3 in photoreceptors strongly inhibited endocytotic uptake of FM1-43.

A local, periactive zone endocytic machinery at photoreceptor synapses in close vicinity to synaptic ribbons.

Photoreceptor ribbon synapses are continuously active synapses with large active zones that contain synaptic ribbons. Synaptic ribbons are anchored to the active zones and are associated with large numbers of synaptic vesicles. The base of the ribbon that is located close to L-type voltage-gated Ca(2+) channels is a hotspot of exocytosis. The continuous exocytosis at the ribbon synapse needs to be balanced by compensatory endocytosis. Recent analyses indicated that vesicle recycling at the synaptic ribbon is also an important determinant of synaptic signaling at the photoreceptor synapse. To get insights into mechanisms of vesicle recycling at the photoreceptor ribbon synapse, we performed super-resolution structured illumination microscopy and immunogold electron microscopy to localize major components of the endocytotic membrane retrieval machinery in the photoreceptor synapse of the mouse retina. We found dynamin, syndapin, amphiphysin, and calcineurin, a regulator of activity-dependent endocytosis, to be highly enriched around the active zone and the synaptic ribbon. We present evidence for two clathrin heavy chain variants in the photoreceptor terminal; one is enriched around the synaptic ribbon, whereas the other is localized in the entry region of the terminal. The focal enrichment of endocytic proteins around the synaptic ribbon is consistent with a focal uptake of endocytic markers at that site. This endocytic activity functionally depends on dynamin. These data propose that the presynaptic periactive zone surrounding the synaptic ribbon complex is a hotspot of endocytosis in photoreceptor ribbon synapses.

Global detection of protein kinase D-dependent phosphorylation events in nocodazole-treated human cells.

Protein kinase D (PKD) is a cytosolic serine/threonine kinase implicated in regulation of several cellular processes such as response to oxidative stress, directed cell migration, invasion, differentiation, and fission of the vesicles at the trans-Golgi network. Its variety of functions must be mediated by numerous substrates; however, only a couple of PKD substrates have been identified so far. Here we perform stable isotope labeling of amino acids in cell culture-based quantitative phosphoproteomic analysis to detect phosphorylation events dependent on PKD1 activity in human cells. We compare relative phosphorylation levels between constitutively active and kinase dead PKD1 strains of HEK293 cells, both treated with nocodazole, a microtubule-depolymerizing reagent that disrupts the Golgi complex and activates PKD1. We identify 124 phosphorylation sites that are significantly down-regulated upon decrease of PKD1 activity and show that the PKD target motif is significantly enriched among down-regulated phosphorylation events, pointing to the presence of direct PKD1 substrates. We further perform PKD1 target motif analysis, showing that a proline residue at position +1 relative to the phosphorylation site serves as an inhibitory cue for PKD1 activity. Among PKD1-dependent phosphorylation events, we detect predominantly proteins with localization at Golgi membranes and function in protein sorting, among them several sorting nexins and members of the insulin-like growth factor 2 receptor pathway. This study presents the first global detection of PKD1-dependent phosphorylation events and provides a wealth of information for functional follow-up of PKD1 activity upon disruption of the Golgi network in human cells.