Identification of the Source of Secreted Proteins in the Kidney by Brefeldin A Injection

Chronic kidney disease (CKD) is one of the top ten leading causes of death in the USA. Acute kidney injury (AKI), while often recoverable, predisposes ...

Vanderbilt University Medical Center

Kidney disease is estimated to affect 15% of the world's population. Autophagy is a key homeostatic pathway in eukaryotic cells, which has been linked to numerous pathological states. In the kidney, autophagy has been shown to modulate both acute and chronic injuries. Despite the importance of autophagy in kidney disease, few techniques to precisely monitor autophagic flux in kidney tissue are available. Here we describe an improved technique to quantify autophagic flux using an RFP-GFP-LC3 reporter mouse and super-resolution microscopy. Using structured illumination microscopy, we can resolve individual autophagosomes within kidney tubular cells. We describe the preparation of slides, staining, imaging and data processing. 3D surface rendering is utilized to categorize and quantify autophagosomes by number, size, fluorescence and autophagic flux in response to ischemia.

Quantifying autophagic flux in kidney tissue using structured illumination microscopy.

Maladaptive proximal tubule (PT) repair has been implicated in kidney fibrosis through induction of cell-cycle arrest at G2/M. We explored the relative importance of the PT DNA damage response (DDR) in kidney fibrosis by genetically inactivating ataxia telangiectasia and Rad3-related (ATR), which is a sensor and upstream initiator of the DDR. In human chronic kidney disease, ATR expression inversely correlates with DNA damage. ATR was upregulated in approximately 70% of Lotus tetragonolobus lectin-positive (LTL+) PT cells in cisplatin-exposed human kidney organoids. Inhibition of ATR resulted in greater PT cell injury in organoids and cultured PT cells. PT-specific Atr-knockout (ATRRPTC-/-) mice exhibited greater kidney function impairment, DNA damage, and fibrosis than did WT mice in response to kidney injury induced by either cisplatin, bilateral ischemia-reperfusion, or unilateral ureteral obstruction. ATRRPTC-/- mice had more cells in the G2/M phase after injury than did WT mice after similar treatments. In conclusion, PT ATR activation is a key component of the DDR, which confers a protective effect mitigating the maladaptive repair and consequent fibrosis that follow kidney injury.

Proximal tubule ATR regulates DNA repair to prevent maladaptive renal injury responses.

Chronic kidney disease (CKD) is a global health problem. CKD patients are at high risk of developing cardiovascular disease (CVD), including coronary artery disease, heart failure and stroke. Several factors invoke a vicious cycle of CKD and CVD, which is referred as to "cardiorenal syndrome". Among these factors, the compounds retained through loss of renal excretion play a pathological role in causing atherosclerosis and CVD. These compounds have been broadly classified as uremic toxins because of their accumulation with declining renal function and cytotoxicity. The major uremic toxins contributing to CVD are asymmetric dimethylarginine (ADMA), advanced glycation endproducts (AGE), and trimethyl amine N-oxide (TMAO). ADMA is linked to CVD through regulation of nitric oxide, reactive oxygen species, and renal anemia. AGE not only directly accumulates in the heart and kidney, but interacts with the receptor for AGE (RAGE), leading to cell damage in CVD. TMAO correlates with a high prevalence of CVD and promotes organ fibrosis by itself. The levels of these and other uremic toxins rise with worsening CKD, inducing multiplicative damage in the heart and kidney. Therefore, a better understanding of uremic toxins has great clinical importance for preventing cardiorenal syndrome. This review highlights the molecular mechanism by which these uremic toxins are implicated in CVD and suggests the possible mutual relationship between them.

Uremic Toxin-Targeting as a Therapeutic Strategy for Preventing Cardiorenal Syndrome.

Chronic kidney disease (CKD) is a public health concern that affects approximately 10% of the global population. CKD is associated with poor outcomes due to high frequencies of comorbidities such as heart failure and cardiovascular disease. Uremic toxins are compounds that are usually filtered and excreted by the kidneys. With the decline of renal function, uremic toxins are accumulated in the systemic circulation and tissues, which hastens the progression of CKD and concomitant comorbidities. Gut microbial dysbiosis, defined as an imbalance of the gut microbial community, is one of the comorbidities of CKD. Meanwhile, gut dysbiosis plays a pathological role in accelerating CKD progression through the production of further uremic toxins in the gastrointestinal tracts. Therefore, the gut-kidney axis has been attracting attention in recent years as a potential therapeutic target for stopping CKD. Trimethylamine N-oxide (TMAO) generated by gut microbiota is linked to the progression of cardiovascular disease and CKD. Also, advanced glycation endproducts (AGEs) not only promote CKD but also cause gut dysbiosis with disruption of the intestinal barrier. This review summarizes the underlying mechanism for how gut microbial dysbiosis promotes kidney injury and highlights the wide-ranging interventions to counter dysbiosis for CKD patients from the view of uremic toxins such as TMAO and AGEs.

Dysbiosis-Related Advanced Glycation Endproducts and Trimethylamine N-Oxide in Chronic Kidney Disease.

A polarized collecting duct (CD), formed from the branching ureteric bud (UB), is a prerequisite for an intact kidney. The small Rho GTPase Rac1 is critical for actin cytoskeletal regulation. We investigated the role of Rac1 in the kidney collecting system by selectively deleting it in mice at the initiation of UB development. The mice exhibited only a mild developmental phenotype; however, with aging, the CD developed a disruption of epithelial integrity and function. Despite intact integrin signaling, Rac1-null CD cells had profound adhesion and polarity abnormalities that were independent of the major downstream Rac1 effector, Pak1. These cells did however have a defect in the WAVE2-Arp2/3 actin nucleation and polymerization apparatus, resulting in actomyosin hyperactivity. The epithelial defects were reversible with direct myosin II inhibition. Furthermore, Rac1 controlled lateral membrane height and overall epithelial morphology by maintaining lateral F-actin and restricting actomyosin. Thus, Rac1 promotes CD epithelial integrity and morphology by restricting actomyosin via Arp2/3-dependent cytoskeletal branching.

Bertha C. Elias

Division of Nephrology and Hypertension,
Department of Medicine,
Division of Nephrology and Hypertension, Department of Medicine

Vanderbilt University Medical Center

Identification of the Source of Secreted Proteins in the Kidney by Brefeldin A Injection

Bertha C. Elias

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Division of Nephrology and Hypertension,Department of Medicine,Division of Nephrology and Hypertension, Department of Medicine

Vanderbilt University Medical Center

Identification of the Source of Secreted Proteins in the Kidney by Brefeldin A Injection

Division of Nephrology and Hypertension,
Department of Medicine,
Division of Nephrology and Hypertension, Department of Medicine