Focal segmental glomerulosclerosis is a kidney disease that is manifested as the nephrotic syndrome. It is often resistant to glucocorticoid therapy and progresses to end-stage renal disease in 50 to 70% of patients. Genetic studies have shown that familial focal segmental glomerulosclerosis is a disease of the podocytes, which are major components of the glomerular filtration barrier. However, the molecular cause in over half the cases of primary focal segmental glomerulosclerosis is unknown, and effective treatments have been elusive. We performed whole-genome linkage analysis followed by high-throughput sequencing of the positive-linkage area in a family with autosomal recessive focal segmental glomerulosclerosis (index family) and sequenced a newly discovered gene in 52 unrelated patients with focal segmental glomerulosclerosis. Immunohistochemical studies were performed on human kidney-biopsy specimens and cultured podocytes. Expression studies in vitro were performed to characterize the functional consequences of the mutations identified. We identified two mutations (A159P and Y695X) in MYO1E, which encodes a nonmuscle class I myosin, myosin 1E (Myo1E). The mutations in MYO1E segregated with focal segmental glomerulosclerosis in two independent pedigrees (the index family and Family 2). Patients were homozygous for the mutations and did not have a response to glucocorticoid therapy. Electron microscopy showed thickening and disorganization of the glomerular basement membrane. Normal expression of Myo1E was documented in control human kidney-biopsy specimens in vivo and in glomerular podocytes in vitro. Transfection studies revealed abnormal subcellular localization and function of the A159P-Myo1E mutant. The Y695X mutation causes loss of calmodulin binding and of the tail domains of Myo1E. MYO1E mutations are associated with childhood-onset, glucocorticoid-resistant focal segmental glomerulosclerosis. Our data provide evidence of a role of Myo1E in podocyte function and the consequent integrity of the glomerular filtration barrier.

Shiga toxin (Stx)-producing E.coli O157:H7 has become a global threat to public health; it is a primary cause of diarrhea-associated hemolytic uremic syndrome (HUS), a disorder of thrombocytopenia, microangiopathic hemolytic anemia, and acute renal failure with thrombi occluding renal microcirculation. In this study, we explored whether Stx triggers complement-dependent microvascular thrombosis in in vitro and in vivo experimental settings of HUS. Stx induced on human microvascular endothelial cell surface the expression of P-selectin, which bound and activated C3 via the alternative pathway, leading to thrombus formation under flow. In the search for mechanisms linking complement activation and thrombosis, we found that exuberant complement activation in response to Stx generated an increased amount of C3a that caused further endothelial P-selectin expression, thrombomodulin (TM) loss, and thrombus formation. In a murine model of HUS obtained by coinjection of Stx2 and LPS and characterized by thrombocytopenia and renal dysfunction, upregulation of glomerular endothelial P-selectin was associated with C3 and fibrin(ogen) deposits, platelet clumps, and reduced TM expression. Treatment with anti–P-selectin Ab limited glomerular C3 accumulation. Factor B-deficient mice after Stx2/LPS exhibited less thrombocytopenia and were protected against glomerular abnormalities and renal function impairment, indicating the involvement of complement activation via the alternative pathway in the glomerular thrombotic process in HUS mice. The functional role of C3a was documented by data showing that glomerular fibrin(ogen), platelet clumps, and TM loss were markedly decreased in HUS mice receiving C3aR antagonist. These results identify Stx-induced complement activation, via P-selectin, as a key mechanism of C3a-dependent microvascular thrombosis in diarrhea-associated HUS.

Sodium-glucose cotransporter 2 (SGLT2) inhibitors have pleiotropic properties beyond blood glucose–lowering effects and modify important nonglycemic pathways, leading to end-organ protection. SGLT2 inhibitors display renoprotective effects in diabetic kidney disease, which creates a rationale for testing the therapeutic potential of this drug class in nondiabetic chronic kidney disease. Here, we have shown that dapagliflozin provided glomerular protection in mice with protein-overload proteinuria induced by bovine serum albumin (BSA), to a similar extent as an ACE inhibitor used as standard therapy for comparison. Dapagliflozin limited proteinuria, glomerular lesions, and podocyte dysfunction and loss. We provide the observation that SGLT2 was expressed in podocytes and upregulated after BSA injections. Through in vitro studies with cultured podocytes loaded with albumin we have identified what we believe to be a novel mechanism of action for SGLT2 inhibitor that directly targets podocytes and relies on the maintenance of actin cytoskeleton architecture. Whether SGLT2 inhibitors represent a possible future therapeutic option for some patients with proteinuric glomerular disease who do not have as yet an effective treatment will require ad hoc clinical studies.

Investigated was the effect of high albumin concentrations on proximal tubular cell expression of fractalkine. Human proximal tubular cells (HK-2) were incubated with human serum albumin (HSA), which induced a dose-dependent increase in fractalkine mRNA associated with increased levels of both membrane-bound and soluble forms of the protein. To evaluate the role of nuclear factor κB (NF-κB) activation in HSA-induced fractalkine mRNA, HK-2 cells were infected with a recombinant adenovirus encoding the natural inhibitor of NF-κB, IkBα; a 43% reduction of fractalkine mRNA levels resulted. Similarly, when cells were infected with the recombinant adenovirus expressing dominant negative mutant of the IkB kinase 2, a 55% inhibition of fractalkine mRNA was achieved. p38 mitogen-activated protein kinase was activated by HSA and was involved in NF-κB–dependent transcription of fractalkine. In kidneys of mice with bovine serum albumin overload proteinuria, fractalkine mRNA levels were 2.3-fold greater than those of controls. Fractalkine expression was also induced in tubular epithelial cells in this model. Anti-CXCR1 antibody treatment limited interstitial accumulation of mononuclear cells. Protein overload is a promoter of fractalkine gene induction mediated by NF-κB and p38 activation in proximal tubular cells. Fractalkine might contribute to direct mononuclear cells into peritubular interstitium and enhance their adhesion property, which in turn would favor inflammation and disease progression. E-mail: gremuzzi@marionegri.it