The chloride channel CFTR is not required for cyst growth in an ADPKD mouse model

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by the development of bilateral renal cysts which enlarge continuously, leading to compression of adjacent intact nephrons. The growing cysts lead to a progressive decline in renal function. Cyst growth is driven by enhanced cell proliferation and chloride secretion into the cyst lumen. Chloride secretion is believed to occur mainly by the cAMP-activated cystic fibrosis transmembrane conductance regulator (CFTR), with some contribution by the calcium-activated chloride channel TMEM16A. However, our previous work suggested TMEM16A as a major factor for renal cyst formation. The contribution of CFTR to cyst formation has never been demonstrated in an adult ADPKD mouse model. We used mice with an inducible tubule-specific Pkd1 knockout, which consistently develop polycystic kidneys upon deletion of Pkd1. Cellular properties, ion currents, and cyst development in these mice were compared with that of mice carrying a co-deletion of Pkd1 and Cftr. Knockout of Cftr did not reveal any significant impact on cyst formation in the ADPKD mouse model. Furthermore, knockout of Cftr did not attenuate the largely augmented cell proliferation observed in Pkd1 knockout kidneys. Patch clamp analysis on primary renal epithelial cells lacking expression of Pkd1 indicated an only marginal contribution of CFTR to whole cell Cl− currents, which were clearly dominated by calcium-activated TMEM16A currents. In conclusion, CFTR does not essentially contribute to renal cyst formation in mice caused by deletion of Pkd1. Enhanced cell proliferation and chloride secretion is caused primarily by upregulation of the calcium-activated chloride channel TMEM16A

Loss of Polycystin-1 causes cAMP-dependent switch from tubule to cyst formation

Autosomal dominant polycystic kidney disease is the most common monogenic disease that causes end-stage renal failure. It primarily results from mutations in the PKD1 gene that encodes for Polycystin-1. How loss of Polycystin-1 translates into bilateral renal cyst development is mostly unknown. cAMP is significantly involved in cyst enlargement but its role in cyst initiation has remained elusive. Deletion of Polycystin-1 in collecting duct cells resulted in a switch from tubule to cyst formation and was accompanied by an increase in cAMP. Pharmacological elevation of cAMP in Polycystin 1-competent cells caused cyst formation, impaired plasticity, nondirectional migration, and mis-orientation, and thus strongly resembled the phenotype of Polycystin-1-deficient cells. Mis-orientation of developing tubule cells in metanephric kidneys upon loss of Polycystin-1 was phenocopied by pharmacological increase of cAMP in wildtype kidneys. In vitro, cAMP impaired tubule formation after capillary-induced injury which was further impaired by loss Polycystin-1

Macrophage migration inhibitory factor is regulated by HIF-1α and cAMP and promotes renal cyst cell proliferation in a macrophage-independent manner

Progressive cyst growth leads to decline of renal function in polycystic kidney disease. Macrophage migration inhibitory factor (MIF) was found to be upregulated in cyst-lining cells in a mouse model of polycystic kidney disease and to promote cyst growth. In addition, MIF can be secreted by tubular cells and may contribute to cyst growth in an autocrine manner. However, the underlying mechanisms leading to induction of MIF in cyst-lining cells remained elusive. Here, we demonstrate that hypoxia-inducible transcription factor (HIF) 1α upregulates MIF in cyst-lining cells in a tubule-specific PKD1 knockout mouse. Pharmacological stabilization of HIF-1α resulted in significant increase of MIF in cyst epithelial cells whereas tubule-specific knockout of HIF-1α prevented MIF upregulation. Identical regulation could be found for ABCA1, which has been shown to act as a transport protein for MIF. Furthermore, we show that MIF and ABCA1 are direct target genes of HIF-1α in human primary tubular cells. Next to HIF-1α and hypoxia, we found MIF being additionally regulated by cAMP which is a strong promotor of cyst growth. In line with these findings, HIF-1α- and cAMP-dependent in vitro cyst growth could be decreased by the MIF-inhibitor ISO-1 which resulted in reduced cyst cell proliferation. In conclusion, HIF-1α and cAMP regulate MIF in primary tubular cells and cyst-lining epithelial cells, and MIF promotes cyst growth in the absence of macrophages. In line with these findings, the MIF inhibitor ISO-1 attenuates HIF-1α- and cAMP-dependent in vitro cyst enlargement. KEY MESSAGES: • MIF is upregulated in cyst-lining cells in a polycystic kidney disease mouse model. • MIF upregulation is mediated by hypoxia-inducible transcription factor (HIF) 1α. • ABCA1, transport protein for MIF, is also regulated by HIF-1α in vitro and in vivo. • MIF is additionally regulated by cAMP, a strong promotor of cyst growth. • MIF-inhibitor ISO-1 reduces HIF-1α- and cAMP-dependent cyst growth

In vitro cyst puncture and injury-induced tubule formation using renal epithelial cells

Summary: Collecting-duct-derived renal epithelial cells switch from tubule to cyst formation; however, the cysts still form tubules after injury of the cyst-lining epithelium. Here, we provide a protocol that describes in vitro cyst growth with focus on glass-capillary-induced cyst wall injury to induce tubule formation. We detail steps for the establishment of the in vitro cyst assay, followed by puncture of the cysts in the collagen matrix. We further describe live imaging and steps to analyze the tubule growth.For complete details on the use and execution of this protocol, please refer to Scholz et al. (2022).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics

Isolated thrombotic microangiopathy of the small intestine in a patient with atypical hemolytic uremic syndrome – a case report

Background Atypical hemolytic uremic syndrome (aHUS) is a rare disease characterized by systemic thrombotic microangiopathy (TMA) reflected by hemolysis, anemia, thrombocytopenia and systemic organ injury. The optimal management of aHUS-patients when undergoing kidney transplantation to prevent recurrence in the allograft is eculizumab, an approved recombinant antibody targeting human complement component C5. Case presentation A 39 year-old woman presented with severe abdominal pain, diarrhea and emesis for 3 days. In her past medical history she had experienced an episode of aHUS leading to end stage renal disease (ESRD) in 2007 and a genetic workup revealed a heterozygous mutation in the membrane cofactor protein gene. In 2014 she underwent cadaveric kidney transplantation. Four years later she had to go back on hemodialysis due to allograft failure following a severe systemic cytomegalovirus infection resulting in transplant failure. At presentation she still received calcineurin-inhibitor therapy and reported subfebrile temperatures and pain projecting over the transplant prior to the current symptoms. A contrast enhanced CT-scan of the abdomen revealed inflammatory wall thickening of the small intestine. Diagnostic endoscopy discovered fresh blood in the small intestine without a clear source of bleeding. Histopathology of the small intestine biopsies showed severe thrombotic microangiopathy. Of note, the patient persistently had no signs of systemic hemolysis. Since the TMA of the small intestine was most likely due to aHUS, eculizumab treatment was initiated which abolished the symptoms. Conclusion Here we report a patient with thrombotic microangiopathy with predominant manifestation in a single organ, the small intestine, due to aHUS with absence of systemic signs and symptoms. aHUS patients usually require a secondary trigger for the disease to manifest. In this case, the trigger may be attributed to the dysfunctional renal transplant, which was subsequently explanted. Histology of the explanted kidney showed severe inflammation due to purulent nephritis and signs of cellular rejection. After nephrectomy, we continued eculizumab therapy until the patient completely recovered. No signs of TMA recurred after discontinuation of eculizumab, further supporting the concept of the renal transplant as the main trigger of TMA of the small intestine in our patient

Kif3a Guides Microtubular Dynamics, Migration and Lumen Formation of MDCK Cells

The microtubular motor Kinesin-2 and its subunit Kif3a are essential for the formation of primary cilia, an organelle implicated in a wide spectrum of developmental abnormalities. Outside cilia, Kinesin-2 mediated transport has been implicated in vesicle and N-cadherin transport, but it is unknown if and how extraciliary Kif3a affects basic cellular functions such as migration or the formation of multicellular structures. Here we show that tetracycline inducible depletion of Kif3a in MDCK cells slows epithelial cell migration. Microtubules at the leading edge of Kif3a depleted cells failed to grow perpendicularly into the leading edge and microtubular dynamics were dampened in Kif3a depleted cells. Loss of Kif3a retarded lateral membrane specification and completely prevented the formation of three-dimensional spheres in collagen. These data uncover that Kif3a regulates the microtubular cytoskeleton in the cell periphery and imply that extra-ciliary Kif3a has an unexpected function in morphogenesis

Nephron-specific knockout of TMEM16A leads to reduced number of glomeruli and albuminuria

TMEM16A is a transmembrane protein from a conserved family of calcium-activated proteins that is highly expressed in the kidney. TMEM16A confers calcium-activated chloride channel activity, which is of importance for various cellular functions in secretory epithelia and involved in secretion-dependent renal cyst growth. However, its specific function in renal physiology has remained elusive so far. Therefore, we generated conditional nephron-specific TMEM16A-knockout mice and found that these animals suffered from albuminuria. Kidney histology demonstrated an intact corticomedullary differentiation and absence of cysts. Electron microscopy showed a normal slit diaphragm. However, the total number of glomeruli and total nephron count was decreased in TMEM16A-knockout animals. At the same time, glomerular diameter was increased, presumably as a result of the hyperfiltration in the remaining glomeruli. TUNEL and PCNA stainings showed increased cell death and increased proliferation. Proximal tubular cilia were intact in young animals, but the number of properly ciliated cells was decreased in older, albuminuric animals. Taken together, our data suggest that TMEM16A may be involved in ureteric bud branching and proper nephron endowment. Loss of TMEM16A resulted in reduced nephron number and, subsequently, albuminuria and tubular damage