Endothelial-specific loss of Krüppel-Like Factor 4 triggers complement-mediated endothelial injury.

Thrombotic microangiopathy (TMA) in the kidney represents the most severe manifestation of kidney microvascular endothelial injury. Despite the source of the inciting event, the diverse clinical forms of kidney TMA share dysregulation of endothelial cell transcripts and complement activation. Here, we show that endothelial-specific knockdown of Krüppel-Like Factor 4 (Klf4)ΔEC, an anti-inflammatory and antithrombotic zinc-finger transcription factor, increases the susceptibility to glomerular endothelial injury and microangiopathy in two genetic murine models that included endothelial nitric oxide synthase knockout mice and aged mice (52 weeks), as well as in a pharmacologic model of TMA using Shiga-toxin 2. In all models, Klf4ΔEC mice exhibit increased pro-thrombotic and pro-inflammatory transcripts, as well as increased complement factors C3 and C5b-9 deposition and histologic features consistent with subacute TMA. Interestingly, complement activation in Klf4ΔEC mice was accompanied by reduced expression of a key KLF4 transcriptional target and membrane bound complement regulatory gene, Cd55. To assess a potential mechanism by which KLF4 might regulate CD55 expression, we performed in silico chromatin immunoprecipitation enrichment analysis of the CD55 promotor and found KLF4 binding sites upstream from the CD55 transcription start site. Using patient-derived kidney biopsy specimens, we found glomerular expression of KLF4 and CD55 was reduced in patients with TMA as compared to control biopsies of the unaffected pole of patient kidneys removed due to kidney cancer. Thus, our data support that endothelial Klf4 is necessary for maintenance of a quiescent glomerular endothelial phenotype and its loss increases susceptibility to complement activation and induction of prothrombotic and pro-inflammatory pathways.

Therapeutic Inhibition of VEGF Signaling and Associated Nephrotoxicities.

Inhibition of vascular endothelial growth factor A (VEGFA)/vascular endothelial growth factor receptor 2 (VEGFR2) signaling is a common therapeutic strategy in oncology, with new drugs continuously in development. In this review, we consider the experimental and clinical evidence behind the diverse nephrotoxicities associated with the inhibition of this pathway. We also review the renal effects of VEGF inhibition's mediation of key downstream signaling pathways, specifically MAPK/ERK1/2, endothelial nitric oxide synthase, and mammalian target of rapamycin (mTOR). Direct VEGFA inhibition via antibody binding or VEGF trap (a soluble decoy receptor) is associated with renal-specific thrombotic microangiopathy (TMA). Reports also indicate that tyrosine kinase inhibition of the VEGF receptors is preferentially associated with glomerulopathies such as minimal change disease and FSGS. Inhibition of the downstream pathway RAF/MAPK/ERK has largely been associated with tubulointerstitial injury. Inhibition of mTOR is most commonly associated with albuminuria and podocyte injury, but has also been linked to renal-specific TMA. In all, we review the experimentally validated mechanisms by which VEGFA-VEGFR2 inhibitors contribute to nephrotoxicity, as well as the wide range of clinical manifestations that have been reported with their use. We also highlight potential avenues for future research to elucidate mechanisms for minimizing nephrotoxicity while maintaining therapeutic efficacy.

Podocyte-Specific Induction of Krüppel-Like Factor 15 Restores Differentiation Markers and Attenuates Kidney Injury in Proteinuric Kidney Disease.

BACKGROUND: Podocyte injury is the hallmark of proteinuric kidney diseases, such as FSGS and minimal change disease, and destabilization of the podocyte's actin cytoskeleton contributes to podocyte dysfunction in many of these conditions. Although agents, such as glucocorticoids and cyclosporin, stabilize the actin cytoskeleton, systemic toxicity hinders chronic use. We previously showed that loss of the kidney-enriched zinc finger transcription factor Krüppel-like factor 15 (KLF15) increases susceptibility to proteinuric kidney disease and attenuates the salutary effects of retinoic acid and glucocorticoids in the podocyte. METHODS: We induced podocyte-specific KLF15 in two proteinuric murine models, HIV-1 transgenic (Tg26) mice and adriamycin (ADR)-induced nephropathy, and used RNA sequencing of isolated glomeruli and subsequent enrichment analysis to investigate pathways mediated by podocyte-specific KLF15 in Tg26 mice. We also explored in cultured human podocytes the potential mediating role of Wilms Tumor 1 (WT1), a transcription factor critical for podocyte differentiation. RESULTS: In Tg26 mice, inducing podocyte-specific KLF15 attenuated podocyte injury, glomerulosclerosis, tubulointerstitial fibrosis, and inflammation, while improving renal function and overall survival; it also attenuated podocyte injury in ADR-treated mice. Enrichment analysis of RNA sequencing from the Tg26 mouse model shows that KLF15 induction activates pathways involved in stabilization of actin cytoskeleton, focal adhesion, and podocyte differentiation. Transcription factor enrichment analysis, with further experimental validation, suggests that KLF15 activity is in part mediated by WT1. CONCLUSIONS: Inducing podocyte-specific KLF15 attenuates kidney injury by directly and indirectly upregulating genes critical for podocyte differentiation, suggesting that KLF15 induction might be a potential strategy for treating proteinuric kidney disease.

Krüppel-Like Factor 15 Mediates Glucocorticoid-Induced Restoration of Podocyte Differentiation Markers.

Podocyte injury is the inciting event in primary glomerulopathies, such as minimal change disease and primary FSGS, and glucocorticoids remain the initial and often, the primary treatment of choice for these glomerulopathies. Because inflammation is not readily apparent in these diseases, understanding the direct effects of glucocorticoids on the podocyte, independent of the immunomodulatory effects, may lead to the identification of targets downstream of glucocorticoids that minimize toxicity without compromising efficacy. Several studies showed that treatment with glucocorticoids restores podocyte differentiation markers and normal ultrastructure and improves cell survival in murine podocytes. We previously determined that Krüppel-like factor 15 (KLF15), a kidney-enriched zinc finger transcription factor, is required for restoring podocyte differentiation markers in mice and human podocytes under cell stress. Here, we show that in vitro treatment with dexamethasone induced a rapid increase of KLF15 expression in human and murine podocytes and enhanced the affinity of glucocorticoid receptor binding to the promoter region of KLF15 In three independent proteinuric murine models, podocyte-specific loss of Klf15 abrogated dexamethasone-induced podocyte recovery. Furthermore, knockdown of KLF15 reduced cell survival and destabilized the actin cytoskeleton in differentiated human podocytes. Conversely, overexpression of KLF15 stabilized the actin cytoskeleton under cell stress in human podocytes. Finally, the level of KLF15 expression in the podocytes and glomeruli from human biopsy specimens correlated with glucocorticoid responsiveness in 35 patients with minimal change disease or primary FSGS. Thus, these studies identify the critical role of KLF15 in mediating the salutary effects of glucocorticoids in the podocyte.

The critical role of Krüppel-like factors in kidney disease.

Krüppel-like factors (KLFs) are a family of zinc-finger transcription factors critical to mammalian embryonic development, regeneration, and human disease. There is emerging evidence that KLFs play a vital role in key physiological processes in the kidney, ranging from maintenance of glomerular filtration barrier to tubulointerstitial inflammation to progression of kidney fibrosis. Seventeen members of the KLF family have been identified, and several have been well characterized in the kidney. Although they may share some overlap in their downstream targets, their structure and function remain distinct. This review highlights our current knowledge of KLFs in the kidney, which includes their pattern of expression and their function in regulating key biological processes. We will also critically examine the currently available literature on KLFs in the kidney and offer some key areas in need of further investigation.

Reduced Krüppel-Like Factor 2 Aggravates Glomerular Endothelial Cell Injury and Kidney Disease in Mice with Unilateral Nephrectomy.

Loss of functional nephrons induces compensatory glomerular hyperfiltration and hypertrophy, leading to the progression of chronic kidney disease. Krüppel-like factor 2 (KLF2), a shear-stress-inducible transcription factor, confers protection against endothelial injury. Because glomerular hyperfiltration is associated with shear stress, we hypothesized that KLF2 may be an important factor in the compensatory response to unilateral nephrectomy (UNX). To test this hypothesis, endothelial cell-specific Klf2 heterozygous knockout mice (KO) and their wild-type littermate control (WT) underwent either UNX or sham-operation. WT-UNX mice developed compensatory renal hypertrophy as expected, whereas KO-UNX mice did not. KO-UNX mice exhibited higher blood pressure, reduced glomerular filtration rate, and significant increase in proteinuria and glomerulosclerosis compared to WT-UNX. Expression of endothelial nitric oxide synthase (official name Nos3), a known transcriptional target gene of KLF2, was significantly reduced and dysregulation of other endothelial genes was also observed in the glomeruli of KO-UNX when compared to WT-UNX and sham-operated mice. Furthermore, both podocyte number and expression of podocyte markers were also significantly reduced in KO-UNX glomeruli, indicating a potential cross talk between glomerular endothelial cells and podocytes. Finally, decreased renal expression of KLF2 in nephrectomy patients was associated with the progression of kidney disease. Taken together, our data demonstrate a protective role of KLF2 against glomerular endothelial cell injury and progression of chronic kidney disease in the model of compensatory renal hypertrophy.

Podocyte-specific KLF4 is required to maintain parietal epithelial cell quiescence in the kidney.

Podocyte loss triggering aberrant activation and proliferation of parietal epithelial cells (PECs) is a central pathogenic event in proliferative glomerulopathies. Podocyte-specific Krüppel-like factor 4 (KLF4), a zinc-finger transcription factor, is essential for maintaining podocyte homeostasis and PEC quiescence. Using mice with podocyte-specific knockdown of Klf4, we conducted glomerular RNA-sequencing, tandem mass spectrometry, and single-nucleus RNA-sequencing to identify cell-specific transcriptional changes that trigger PEC activation due to podocyte loss. Integration with in silico chromatin immunoprecipitation identified key ligand-receptor interactions, such as fibronectin 1 (FN1)­αVß6, between podocytes and PECs dependent on KLF4 and downstream signal transducer and activator of transcription 3 (STAT3) signaling. Knockdown of Itgb6 in PECs attenuated PEC activation. Additionally, podocyte-specific induction of human KLF4 or pharmacological inhibition of downstream STAT3 activation reduced FN1 and integrin ß 6 (ITGB6) expression and mitigated podocyte loss and PEC activation in mice. Targeting podocyte-PEC crosstalk might be a critical therapeutic strategy in proliferative glomerulopathies.

Krüppel-like factor 4 is a negative regulator of STAT3-induced glomerular epithelial cell proliferation.

Pathologic glomerular epithelial cell (GEC) hyperplasia is characteristic of both rapidly progressive glomerulonephritis (RPGN) and subtypes of focal segmental glomerulosclerosis (FSGS). Although initial podocyte injury resulting in activation of STAT3 signals GEC proliferation in both diseases, mechanisms regulating this are unknown. Here, we show that the loss of Krüppel-like factor 4 (KLF4), a zinc-finger transcription factor, enhances GEC proliferation in both RPGN and FSGS due to dysregulated STAT3 signaling. We observed that podocyte-specific knockdown of Klf4 (C57BL/6J) increased STAT3 signaling and exacerbated crescent formation after nephrotoxic serum treatment. Interestingly, podocyte-specific knockdown of Klf4 in the FVB/N background alone was sufficient to activate STAT3 signaling, resulting in FSGS with extracapillary proliferation, as well as renal failure and reduced survival. In cultured podocytes, loss of KLF4 resulted in STAT3 activation and cell-cycle reentry, leading to mitotic catastrophe. This triggered IL-6 release into the supernatant, which activated STAT3 signaling in parietal epithelial cells. Conversely, either restoration of KLF4 expression or inhibition of STAT3 signaling improved survival in KLF4-knockdown podocytes. Finally, human kidney biopsy specimens with RPGN exhibited reduced KLF4 expression with a concomitant increase in phospho-STAT3 expression as compared with controls. Collectively, these results suggest the essential role of KLF4/STAT3 signaling in podocyte injury and its regulation of aberrant GEC proliferation.

5 Years Experience With Drug Eluting and Bare Metal Stents as Primary Intervention in Transplant Renal Artery Stenosis.

BACKGROUND: Transplant renal artery stenosis (TRAS) is a common vascular complication after kidney transplantation and is associated with refractory hypertension, volume overload, and graft injury or loss. This article describes 5-year outcomes of endovascular intervention for TRAS with bare metal and drug eluting stents (DES). METHODS: We investigated, as a prospective cohort study, patient and graft outcomes after the targeted use of DES for vessel diameter less than 5 mm and bare metal stents (BMS) for vessel diameter greater than 5 mm as the primary management for TRAS. RESULTS: From March 2008 to November 2014, 57 patients were stented for hemodynamically significant TRAS; 29 received DES, 26 received BMS, and 2 patients received both stent types. They were followed up for a mean of 35.1 ± 22.8 months; a subset of these patients who all received DES were followed up for 61.7 ± 17.5 months. Mean serum creatinine declined from 2.87 ± 1.5 mg/dL at the time of intervention to 1.98 ± 0.76 mg/dL (P < 0.001) at one month follow-up and was 1.96 ±0.92 mg/dL (P < 0.001) at 35.1 ± 22.8 months. Mean systolic blood pressure declined from 159.05 ± 19.68 mm Hg at time of intervention to 135.65 ± 15.10 mm Hg (P < 0.001) at most recent visit. Clinically driven restenosis requiring repeat revascularization occurred in 15.7% of patients. CONCLUSIONS: Primary stenting with DES and BMS is both successful in the initial treatment of TRAS and also produced an immediate and long-term reduction in serum creatinine and systolic blood pressure.

Different Management Options for Transplant Ureteral Obstructions within an Inguinal Hernia.

Ureteral obstruction secondary to an inguinal hernia with transplant ureteral component is an extremely rare entity with only several case reports found in literature. In all previously reported cases, management of the obstruction involved temporary drainage with ureteral stenting or nephrostomy tube placements followed by delayed definitive repair. We present two case reports, here one being the first one managed by immediate definitive repair via ureteral reimplant and herniorrhaphy and a second case by delayed definitive repair after percutaneous nephrostomy tube placement. Both patients continued to do well postoperatively with normalization of renal function on follow-up.

Triple Diuretics and Aquaretic Strategy for Acute Decompensated Heart Failure due to Volume Overload.

Diuretics, including furosemide, metolazone, and spironolactone, have historically been the mainstay of therapy for acute decompensated heart failure patients. The addition of an aquaretic-like vasopressin antagonist may enhance diuresis further. However, clinical experience with this quadruple combination is lacking in the acute setting. We present two hospitalized patients with acute decompensated heart failure due to massive fluid overload treated with a combination strategy of triple diuretics in conjunction with the aquaretic tolvaptan. The first patient lost 72.1 lbs. (32.7 kg) with an average urine output of 3.5 to 7.5 L/day over eight days on combined therapy with furosemide, metolazone, spironolactone, and tolvaptan. The second patient similarly achieved a weight loss of 28.2 lbs. (12.8 kg) over 4 days on the same treatment. Both patients maintained stable serum sodium, potassium, and creatinine over this period and remained out of the hospital for more than 30 days. Thus, patients hospitalized with acute decompensated heart failure due to volume overload can achieve euvolemia rapidly and without electrolytes disturbances using this regimen, while being under the close supervision of a team of cardiologists and nephrologists. Additionally, this therapy can potentially decrease the need for ultrafiltration and the length of hospital stay.