Genetic prescreening of a candidate for laser refractive surgery identifies risk for inadequate tissue response: a case report

BACKGROUND: Inadequate response to corneal laser refractive surgery, e.g., ectatic corneal diseases, may not be identified by conventional examinations, hence creating therapeutic uncertainty. Herein we demonstrate the application of genetic prescreening to augment preassessment for corneal laser refractive surgery and highlight the ability to prevent the possibility of enrolling a subject at risk for developing ectatic corneal diseases. CASE PRESENTATION: Preoperative tests were performed alongside deoxyribonucleic acid (DNA) sequencing of 75 genes specific to the structure and health of the eye of a 44-year-old Caucasian male candidate for corneal laser refractive surgery. The patient had no medical, family, or psychosocial history, nor symptoms that could lead to suspect any corneal abnormalities, and conventional preoperative tests confirmed that no corneal abnormalities were present. The sequencing results uncovered rare DNA variants within the ADGRV1, PTK2, ZNF469, and KRT15 genes. These variants were considered potential risk factors for inadequate response in the patient post corneal laser refractive surgery. Subsequent reevaluation with three different last-generation corneal tomographers identified in the left eye a “warning” for a deformity of the posterior profile of the cornea. CONCLUSIONS: Genetic prescreening identifies potential risk of inadequate response to corneal laser refractive surgery where current technologies in use may lead to a hazardous predictive diagnostic uncertainty

Dysregulation of Cationic Channels in Chronic Kidney Diseases

According to the Centers for Disease Control and Prevention, one in 10 American adults, has some level of chronic kidney disease (CKD), a condition characterized by reduced kidney function over time. Although recent research has uncovered many pathways and mechanisms involved in the pathophysiology of kidney diseases, this has not yet led to development of new drugs for the treatment of patients with these conditions. In this dissertation, we introduce two potential therapeutic targets for different forms of CKD. First, we discuss gating properties of the transient receptor potential cationic-6 (TRPC6) channel, then we show dysregulation of this channel in models of focal segmental glomerulosclerosis (FSGS). In a separate chapter, we introduce another channel protein, the N-methyl-D-aspartate (NMDA) receptor, as a potential therapeutic target for treatment of diabetic nephropathy. Much of this work entailed making whole-cell recordings from highly specialized kidney cells called podocytes. This technique was used to measure TRPC6 channel activity in cells in vitro, as well as in ex vivo preparations in which podocytes are still attached to the isolated glomerular capillary. Serum samples from a variety of primary FSGS patient groups were obtained from collaborators. We used the sera to treat our cells in vitro and investigate the effect of soluble factors in the patients’ serum on the activity and expression levels of TRPC6 channels. Regarding the other target, we studied the effect of NMDA inhibitors in alleviating the development of diabetic nephropathy in vivo. I concluded that TRPC6 channels are dysregulated in FSGS. This suggested that further development of TRPC6 inhibitors might be warranted as potential therapeutic agents. We observed that diabetes caused a marked increase in the expression of renal NMDA receptors, and that sustained treatment with NMDA antagonists reduces the progression of nephropathy in two mouse models of type-1 diabetes. Consequently, it is possible that this class of drugs can be useful for reducing the progression of nephropathy.Biology and Biochemistry, Department o

RETRACTED: Pleiotropic signaling evoked by tumor necrosis factor in podocytes (Retracted article. See vol. 310, pg. F1423, 2016)

TNF has been implicated in glomerular diseases, but its actions on podocytes are not well understood. Endogenous TNF expression is markedly increased in mouse podocytes exposed to sera from patients with recurrent focal segmental glomerulosclerosis, and TNF is able to increase its own expression in these cells. Exposure of podocytes to TNF increased phosphorylation of NF-kappa B p65-RelA followed by increased tyrosine phosphorylation of STAT3. STAT3 activation was blocked by the NF-kappa B inhibitor JSH-23 and by the STAT3 inhibitor stattic, whereas TNF-evoked NF-kappa B activation was not affected by stattic. TNF treatment increased nuclear accumulation of nuclear factor of activated T cells (NFAT) c1 in podocytes, a process that occurred downstream of STAT3 activation. TNF also increased expression of cyclin D1 but had no effect on cyclin-dependent kinase 4, p27(kip), or podocin. Despite its effects on cyclin D1, TNF treatment for up to 72 h did not cause podocytes to reenter the cell cycle. TNF increased total expression of transient receptor potential (TRP)C6 channels through a pathway dependent on NFATc1 and increased the steady-state expression of TRPC6 subunits on the podocyte cell surface. TNF effects on TRPC6 trafficking required ROS. Consistent with this, La3+-sensitive cationic currents activated by a diacylglycerol analog were increased in TNF-treated cells. The effects of TNF on NFATc1 and TRPC6 expression were blocked by cyclosporine A but were not blocked by the pan-TRP inhibitor SKF-96365. TNF therefore influences multiple pathways previously implicated in podocyte pathophysiology and is likely to sensitize these cells to other insults

APOL1 renal risk variants promote cholesterol accumulation in tissues and cultured macrophages from APOL1 transgenic mice

Apolipoprotein L1 (APOL1) genetic variants G1 and G2, compared to the common allele G0, are major risk factors for non-diabetic kidney disease in African descent populations. APOL1 is a minor protein component of HDL, as well as being expressed in podocytes and vascular cells. Reverse cholesterol transport involves the transport of cholesterol to HDL by cellular ATP-binding cassette; ABCA1 and ABCG1 with subsequent delivery from peripheral tissues to the liver. With impaired reverse cholesterol transport, lipid accumulation occurs and macrophages morphologically transform into foam cells, releasing inflammatory factors. We asked whether the APOL1 risk variants alter peripheral cholesterol metabolism and specifically affect macrophage cholesterol efflux. Tissues and bone marrow (BM)-derived monocytes were isolated from wild-type mice (WT) and from BAC/APOL1 transgenic (APOL1-G0, APOL1-G1, and APOL1-G2) mice, which carry a bacterial artificial chromosome that contains the human APOL1 genomic region. Monocytes were differentiated into macrophages using M-CSF, and then polarized into M1 and M2 macrophages. Cholesterol content, cholesterol efflux, and ABCA1 and ABCG1 mRNA expression were measured. Kidney, spleen, and bone marrow-derived macrophages from APOL1-G1 and -G2 mice showed increased cholesterol accumulation and decreased ABCA1 and ABCG1 mRNA levels. BM-derived macrophages from APOL1-G1 and -G2 mice showed significantly reduced cholesterol efflux compared to WT or APOL1-G0 macrophages. Taken together, the evidence suggests that APOL1-G1 and -G2 risk variants impaired reverse cholesterol transport through decreased expression of cholesterol efflux transporters suggesting a possible mechanism to promote macrophage foam cell formation, driving inflammation in the glomerulus and renal interstitium