Wednesday, July 23, 2014

Hyperammonemia - When should we start dialysis?

I would like to discuss a case that I recently saw in renal consult. He was a man in his 60s with history of end stage liver disease who received a liver transplant. His hospital course was complicated by anuric ATN and liver graft failure. As a result, he was started on dialysis on post-operative day 0.  Dialysis was stopped on post-op day 2 due to recovering renal function. On post-op day 3 he became encephalopathic. His ammonia level was elevated to 337 and did not improve with conventional therapy with lactulose/rifaximin. The question was whether to start dialysis or not in spite of his recovering renal function.

Causes: The urea cycle in the liver in which ammonia gets converted to urea is responsible for excretion of waste nitrogen.  Hyperammonemia in newborns is most commonly associated with inherited disorders of amino acid and organic acid metabolism. Causes in adults include Reye’s syndrome, liver failure, sepsis especially infections with urea splitting organisms, high dose chemotherapy, drugs (salicyclates, valproate), gastrointestinal bleeding, multiple myeloma, parenteral nutrition and late onset of urea cycle defects. The latter usually presents with episodic encephalopathy precipitated by metabolic stressors like infection, anesthesia or pregnancy.

Clinical features: Hyperammonemia can be life threatening and if persistent can lead to irreversible neuronal damage. It leads to cerebral edema causing progressive encephalopathy. Respiratory alkalosis is common due to central hyperventilation. Severe hyperammonemia can also cause seizures. An MRI brain is usually consistent with hypoxic ischemic encephalopathy

When to start dialysis? There are no published guidelines for when to initiate dialysis in a patient with hyperammonemia due to urea cycle defects. It is commonly indicated if the ammonia blood level is greater than three to four times the upper limit of normal or greater than 200 micromoles/L. Continuous hemodialysis is started with higher flow rates and is the most effective treatment in rapidly reducing ammonia levels.  Even though ammonia is osmotically active, the rapid removal of ammonia is not associated with disequilibrium syndrome mainly due to two reasons: First, there is a rapid equilibration of ammonia across the cell membrane. Secondly, the total amount in the blood, even in severe hyperammonemia, is only about 200 micromoles. This contributes less than 1 mosm per liter to total osmolality and therefore, even if it were all removed at once, the change in osmolality is too small to make cause disequilibrium. Contrast this with ammonia levels in the urine which are typically in the millimolar range.

The question remains whether to start dialysis in the setting of acute severe hyperammonemia (levels > 200 micromoles/L) and encephalopathy in adults with liver failure and normal kidney function. I was not able to find any literature on it and would like to know what the practice in other institutions is?  I believe since severe hyperammonemia can lead to irreversible brain damage, dialysis should be instituted. See this previous post concerning hyperammonemia in individuals with myeloma.

Posted by Silvi Shah

Tuesday, July 22, 2014

Tick borne diseases for the nephrologist - Babesiosis

The following few posts are a summary of tick borne illnesses that can cause renal failure and/or electrolyte problems. 

First identified on Nantucket Island in 1969 and was initially know as Nantucket fever.

Endemic areas.
CDC map. Reported cases in 2012
Cases have been reported in Europe (Croatia, France, Great Britain, Ireland, Portugal, Spain, Sweden, Switzerland)(Babesia divergens).

The tick.
Ixodes scapularis. The Blacklegged tick.

The pathogen.
Babesia microti and B. duncani (USA). B divergens (Europe). Protozoan parasites infecting red blood cells
Humans are not a natural host. Infection by blood transfusion has been reported.

Incubation period 1 – 9 weeks

Clinical features.
Fever, chills, sweats, Malaise, fatigue, Myalgia, arthralgia, headache, Gastrointestinal symptoms, such as anorexia and nausea (less common: abdominal pain, vomiting)
Dark urine
Less common: cough, sore throat, emotional lability, depression, photophobia, conjunctival injection
Mild splenomegaly, mild hepatomegaly, or jaundice may occur in some patients

Lab features
Haemolytic anemia, thrombocytopenia, renal failure, transaminitis.

Diagnosis - Light microscopy of blood cells, serology and PCR for B. microti or B. duncani

Treatment - Atovaquone plus azithromycin or quinine plus clindamycin orally for 7 to 10 days.
Atovaquone plus azithromycin is preferred as this combination is better tolerated.

The source for this review is mainly from the CDC website as well as various references cited in the posts. The tick pictures provided may differ from what might be found on a bitten human. Ticks become larger and engorged after feeding and will look different.

Thursday, July 17, 2014

The Lone Star Tick

Recently a 65-year-old female was referred to an ED in the state of Missouri complaining of fevers, chills, headache, diarrhea and vomiting occurring over the last week. 3 weeks prior she had received an orthotopic liver transplant. Her post transplant course was unremarkable and she was discharged on prograf 2mf bid, myfortic 360mg bid, prednisone tapering, Bactrim single strength daily and valcyte 450mg od. She also took thyroid replacement, Januvia, warfarin and aspirin. Past medical history included diabetes, heart failure, dysfunctional uterine bleeding, hypothyroid and PUD. She also had CKD with a creatinine about 1.4mg/dl. On examination her Temp was 38.6, HR 110, BP 113/76 and O2sats 99% on RA. She had no nuchal rigidity but reported a sore neck. She had an erythematous area on her lower back. The rest of her exam was normal.

Her initial labs were:
Hb 6.3, WCC 2.7 (94%neuts, 5.8%lymphs), platelets 35
NA 122, K 5.6, Cl 100, CO2 11, BUN36, Creat 2.3, Gluc 173
Arterial pH 7.31, CO2 20, O2 102
AST 52, ALT 41, AP 155, GGT 171, Bili 0.6, Alb 3.3
UA, No blood, 1+protein, 2 rbc, 2 wbc

She had a normal CT brain and was started on multiple antibiotics in the ED.

On further questioning her daughter reported removing a tick from her back after the patient went out looking at deer close to her house.
Lets pretend the daughter brought in a picture of the tick (see above)!

Lab trends; admission to discharge.
Hb 6.3
WCC 2.7                   1.4           0.6           0.5           0.3           1.2           2.4
94%neut                90            91            87            83            60            59
5.8%lym                 6               6.7           9.2           14            16            28
plt 35                       16            18            24            28            26            31
NA 122                    128         132         135
K 5.6                   
Cl 100
Co2 11
Creat 2.3                 2.2           1.8           1.6                                               1.3
Gluc 173
pH 7.31
CO2 20
O2 102
AST 52                                                                                                               31
ALT 41                                                                                                               31
AP 155                                                                                                              249
GGT 171                                                                                                                            
Bili 0.6                                                                                                               0.8
Alb 3.3
No blood
2 rbc
2 wbc
No lumbar puncture was performed.

To summarize, this lady had constitutional symptoms, fever, neurological symptoms, GI symptoms with a rash and a tick bite. She had pancytopenia, transaminitis, hyponatreamia and renal failure.

Her antiproliferative medication and anti-infective medications were held (Myfortic, valcyte and Bactrim).
IV Doxycycline was commenced.
Sodium and creatinine improved and potassium became low, likely due to GI losses.
Interestingly her leucopenia followed the classical pattern of lymphopenia followed by leucopenia and she required G-CSF. Her transaminases were normal at the time of discharge.

Diagnosis = Ehrlichiosis 

The first case of human ehrlichiosis was described in 1986.
The two most important species to infect humans are Ehrlichia chaffeensis which causes human monocytic ehrlichiosis (HME) and Anaplasma phagocytophilum which causes human granulocytic anaplasmosis (HGA). Both of these diseases have the same vector and have very similar clinical and laboratory features. Ehrlichia ewingii is a less common cause of ehrlichiosis than Ehrlichia chaffeensis.
Ehrlichiae are obligate intracellular bacteria found in membrane bound vacuoles in human and animal leukocytes.

The most endemic area is the southeastern USA – ‘the tick belt’. See the CDC map for the endemic regions in the USA. Cases have also been reported in Europe, Africa, South America and Mexico.

The lone star tick (Amblyomma americanum) is recognized by the CDC as the principal vector of Ehrlichia chaffeensis and Ehrlichia ewingii in the U.S.; both disease agents are responsible for causing ehrlichiosis in humans. White-tailed deer are a primary host of the lone star tick and appear to serve as a natural reservoir for E. chaffeensis. The lone star tick is also a vector of Francisella tularensis, causal agent of tularemia. Adult ticks parasistize deer, cattle, horses, feral swine, sheep, dogs, and humans.

Most infections occur in the spring and summer in the USA.

The clinical manifestations in the elderly and immunosuppressed can be very severe but the following are the usual clinical features:

Fever - Some fevers can be protracted over weeks
Malaise, myalgia, headache and chills – 2/3
Nausea, vomiting and arthralgia – ¼ to ½
Rash (Macular, mucopapular, petechial) – 1/3
Meningism – ¼

More rarely – Seizures, coma, renal failure, heart failure and respiratory failure
There has been a single case of myocarditis and multi-organ failure in a healthy adolescent.

Laboratory findings:
Most common triad is leucopenia, thrombocytopenia and elevated transaminases.

Leucopenia. This tends to be caused by lymphopenia initially followed by neutropenia as in this patient.

Elevated transaminases, LDH and Alk phos
Elevated creatinine

CSF, when neurological symptoms
Lymphocytic pleocytosis and elevated CSF protein

Usually by PCR methods. Note this test may not detect the recently reported third species, E. muris, found in Wisconsin and Minnesota.

Differential diagnosis
This can be a difficult diagnosis to make. It is clinically and geographically similar to RMSF. It can also present like mononucleosis, TTP, hematologic malignancy, cholangitis, the early phases of hepatitis A infection. This is especially so in immunocompromised patients whose clinical features may not be as obvious initially. Common transplant drugs such as Bactrim/Septra, valganciclovir, mycophenolate and azathioprine can also cause cytopenias.

Treatment (adults)
Doxycycline 100mg iv or po bid for about 10 days.
Note this will also treat RMSF which is often confused with ehrlichiosis.

Outcomes. Mortality is about 5%. Most commonly due to viral or fungal super-infections (Invasive aspergillosis, candida, HSV).

This interesting case illustrates the difficulty in diagnosing tick borne infections. They can be lethal and severe in our immunosuppressed transplant population and can be a cause of renal failure in any patient. Also of interest in this case is the lymphocytopenic and neutropenic trending that is usually more peculiar to Anaplasma phagocytophilum which causes human granulocytic anaplasmosis (HGA). We did not test for A phagocytophilum as ehrlichae PCR was positive.


A bit of housekeeping here - I noticed that all of our current course announcements were out of date. These have now been updated with the most recent versions of each course including the following courses coming up shortly:

ASN Board Review Course - August 2-8 2014
HD University - August 15-16 2014
Brigham Renal Board Review Course - August 4-8 2014
Medstar Kidney and Pancreas Transplantation Course - October 11, 2014
Ultrasonography for Nephrologists - October 25-26 2014 (which I recently attended and thought was excellent)

All of these courses have substantially reduced fees for trainees.

Here is the list of courses

Edit: Just to mention that the director of the ISHD meeting, John Daugirdas, contacted me and let me know that the new edition of the Handbook of Dialysis will be out later this year. If you were thinking of buying it (and I did in the past), you might want to wait until then.

Wednesday, July 16, 2014

Baclofen in dialysis patients: Just say no!

At our hospital, we started noticing a pattern of admissions among some of our maintenance dialysis patients. In one case, an elderly woman with DM, peripheral arterial disease, and ESRD on thrice weekly hemodialysis was sent to the emergency department from her nursing facility for altered mental status. She was normal three days prior to admission, but when her nurse found her confused and difficult to arouse, they sent her to our ED. We got consulted to continue maintenance dialysis, but we noticed that she had a fluent aphasia and marked perseveration. The rest of her exam and workup was unremarkable.  I immediately called the nursing facility and had them fax over her medication administration record. Lo and behold, three days prior she was started on baclofen 5mg three times daily for complaints of lower extremity leg pain (which was probably her claudication anyway). We diagnosed her with acute baclofen neurotoxicity.

Baclofen is an oral antispasmodic that is used to treat muscle spasticity. Chemically, it is very similar to the CNS neurotransmitter GABA and it acts as an agonist at GABAB receptors, resulting in an inhibitory effect on neurons.  Baclofen is rapidly absorbed after ingestion and 90% of the drug is excreted unchanged by glomerular filtration. Therefore, patients with CKD and ESRD are at significant risk from baclofen accumulation and toxicity. The most common complaints are non-specific: drowsiness, headache, lethargy, nausea or vomiting. However, with a severe overdose, profound CNS depression occurs with respiratory depression, bradycardia, hypotonia, areflexia, myoclonus, or seizure activity.

The importance of baclofen toxicity in patients with renal failure has only recently been described and remains unappreciated by many physicians. El-Husseini et al. compiled a nice series of 41 cases of baclofen toxicity in patients with renal insufficiency. The majority of the patients had ESRD (62.9%) and the remaining either had advanced CKD or AKI. Despite being a small, heterogeneous case series, a few trends were seen. The onset of symptoms typically came two to four days after initiating the drug, and the mean daily dose was 20mg per day.

Since baclofen is small molecule (213 Daltons) and it has a relatively low volume of distribution and low degree of protein binding, it is readily removed by hemodialysis. There are only a few of clearance studies out there, but one report found that 4 hours of HD with a high-flux membrane resulted in a clearance of 120 ml/min, equivalent to a normal GFR! Since we have been more aware of baclofen, we have discovered more cases of neurotoxicity in our ESRD patients. We treat with daily sessions of high-flux HD for at least 4 hours until symptoms disappear. Our patient returned to her baseline after 3 consecutive days of HD. The authors of the case series highlight the lack of official dosing guidelines for baclofen in patients with renal insufficiency. Thus, many providers are unaware that this drug is contraindicated in those with advanced CKD and ESRD. Renal fellows are already primed to check for excessive gabapentin doses and NSAIDS, but I would add Baclofen to the list of drugs that should not be given to any patient with advanced CKD or ESRD.

Posted by John Roberts

Tuesday, July 15, 2014

Lithium and the Kidney: Old Observations & New Insights

Lithium is indispensable as an effective treatment for bipolar affective disorder. However, it has a narrow therapeutic index with desired therapeutic levels between 0.6 and 1.2 mEq/L. Lithium is handled by the kidney (responsible for almost all its excretion) in a manner very similar to sodium. It has a molecular weight of 7 daltons, has a moderate volume of distribution, is <10% protein bound and is therefore readily dialyzable. Lithium may cause of myriad of renal related toxicities, some of which are classic descriptions and some which are less well known. Much of this has been discussed on RFN before (here, here) but I felt a refresher with some additions was due.

Nephrogenic Diabetes Insipidus (NDI)

This is perhaps the best know and most common complication of lithium therapy, with an estimated prevalence of 20-70%. Patients present with polyuria and polydipsia due to a urinary concentrating defect that can lead to volume depletion, especially if access to free water is restricted. Lithium downregulates aquaporin-2, vasopressin-activated water channels expressed on the collecting duct principal cells. It is worth noting that ongoing volume depletion from NDI may aggravate the risk of supra-therapeutic levels and therefore chronic nephrotoxicity. Amiloride may help as it inhibits lithium reabsorption via ENaC in the collecting duct.    

Renal tubular Acidosis (RTA)

Lithium may induce a hyperchloremic non-anion gap metabolic acidosis, analogous to a distal RTA. It is likely due to decreased distal proton secretion. Unlike acidosis associated with amiloride, it is not associated with hyperkalemia probably because of their different effects on distal H+/K+-ATPase function.

Acute Lithium Nephrotoxicity

This may occur as an overdose in newly treated patients or those on long-term lithium therapy. Acute lithium intoxication may cause altered mental status and acute kidney injury. Drugs which decrease GFR (NSAIDs/RAAS inhibitors) may exacerbate intoxication as can thiazides, which cause a natriuresis and a subsequent reabsorption of Na (and lithium). With preserved renal function and mild intoxication, increasing urine output/forced diuresis may be all that is needed. Care should be taken using 0.9%NaCl if hypernatremia from NDI is present. As mentioned above, lithium is readily dialyzable and hemodialysis (ideally using a high flux membrane to aid clearance) is the primary management for severe cases. A lithium level >4mEq/L is considered an absolute indication for dialysis in most cases. A level >2.5mEq/L with severe symptoms, or renal impairment which will slow native clearance, is also an indication. If in doubt, most nephrologists would have a low threshold for dialysis, although it is usually not needed with a level <2.5mEq/L. Drug levels drop rapidly during dialysis but they do rebound so extended and repeated sessions are usually necessary. Continuous therapies are less efficient so are not desirable if severe intoxication is present but may be used if conventional dialysis is not immediately available.

Chronic Tubulointerstitial Nephritis (TIN)

A chronic TIN with interstitial fibrosis and tubular dropout is the commonest pathology seen when patients with lithium and renal impairment are biopsied. Consistent with this is a bland urine sediment with little/no proteinuria being common (although not exclusive-see next section). As nephrologists, our view of the prevalence of lithium induced CKD is skewed. The exact prevalence is difficult to determine but many patients have preserved renal function. The tricky decision is whether (and if so when) to stop therapy? This is rarely a decision the nephrologist can make alone and should be balanced with efficacy of treatment/alternatives available and usually led by the psychiatrist in my opinion.

Nephrotic Syndrome

It is less well known that lithium may be associated with a glomerulopathy. Minimal change disease is most often described but membranous nephropathy and FSGS has also been reported. Cases were considered lithium induced as proteinuria disappeared upon cessation of the drug, and when re-challenged (for psychiatric reasons), the nephrotic syndrome recurred. A series of 24 patients with lithium-induced nephrotoxicity from New York included one quarter with nephrotic-range proteinuria. My initial thoughts were that the FSGS lesions were likely secondary to nephron loss and resulting hyperfiltration injury. However, arguing against this is that presence of FSGS lesions did not correlate with severity of tubulointerstitial lesions. Moreover, there was a high incidence of extensive podocyte foot process effacement, to a degree uncommon in secondary FSGS. The mechanism of glomerular toxicity is unclear.

Renal Cell Tumors

It has recently been reported in Kidney International that chronic lithium use is associated with an increased risk of kidney tumours.  After a mean duration of lithium exposure of >21 years, patients had a significantly higher risk of solid renal tumours compared with gender, age and eGFR matched controls [Standardized Incidence Ratio i.e. ratio of observed-to-expected numbers of renal cancers was 7.51 and 13.69 in men and women respectively]. The tumours were a mixed bag of benign (oncocytomas, angiomyolipomas etc.) and malignant (clear cell, stromal, papillary) lesions. While these results are thought provoking, this was a retrospective study and the design seems open to detection bias in my opinion (were patients on lithium screened more than regular individuals?).


As previously posted, lithium may cause hypercalcemia and stimulate PTH via a variety of postulated mechanisms (see previous post).

Friday, July 11, 2014

Parvovirus B19 and the Kidney

Parvovirus B19 (PVB19) is a small, non-enveloped, single-stranded DNA virus belonging to the Erythrovirus genus, named for their tropism for erythroid precursor cells. It is the only known parvovirus to infect humans. By late adulthood, most people have IgG Anti-PVB19 serology demonstrating previous exposure, often from an asymptomatic infection. However, infection with PVB19 may cause a variety of clinical syndromes including fifth disease (‘Erythema infectiosum‘, a childhood viral exanthem), a polyarthropathy, pure red cell aplasia and hydrops fetalis in utero. The nephrologist may encounter PVB19 in 3 broad settings: 


The incidence of PVB19 infection post kidney transplantation is hard to accurately determine but is likely in the range of 1-10%. Infection tends to occur in the first year and frequently in the first few weeks, suggesting possible donor transmission but the mechanism of infection/trans mission is unknown. Clinical syndromes are mainly acute anemia and chronic pure red cell aplasia, although a pancytopenia may develop. The few cases I have seen have been easy to identify, as patient presented with a profound isolated anemia. The diagnosis of chronic anemia or pancytopenia may be more protracted as these are obviously common post-transplant complications (graft dysfunction; drugs-immunosuppressants, anti-virals, co-trimoxazole, ACE inhibitor; other infections-CMV etc.). Treatment consists of reducing immunosuppression (usually the anti-metabolite) and IVIg. Similar to other viral infections (CMV, Polyoma viruses), there is some thought that PVB19 may be associated with allograft dysfunction or even acute rejection (?injured endothelium exposing previously hidden epitopes; no hard evidence for this however). A case series of thrombotic microangiopathy with PVB19 infection post transplant isolated PVB19 DNA from graft biopsies, however, overall the evidence for PVB19-induced allograft dysfunction is weak at present. 

Glomerular Disease 

PVB19 may cause a variety of glomerular lesions in immunocompetent hosts. These were first noted as associations with PVB19 viremia, however, subsequently viral DNA has been identified in renal cells from biopsy tissue. The most well described pattern of injury is FSGS, particular a collapsing glomerulopathy (see image), where the PVB19 genome has been detected in podocytes and parietal epithelial cells by PCR. Proliferative glomerulonephritis has also been temporally associated with PVB19 infection. A syndrome similar to acute post-infectious glomerulonephritis may occur, displaying hypocomplementemia, endocapillary and mesangial proliferation with subendothelial electron dense deposits. Acute glomerulonephritis may be more common in patients with sickle cell disease after an aplastic crisis due to PVB19. Given the ubiquitous nature of PVB19, it is likely that host factors contribute to why certain individuals manifest glomerular disease, with genetic factors, as always, being implicated. Viral DNA may be found in renal tissue after the acute infection has passed and the glomerular lesions will not necessarily improve with resolution of the infection. 

Dialysis Patients 

Patient with ESRD are considered to be at risk for aplastic crisis due to PVB19, albeit with a paucity of data to support that claim. The presence of abundant immature erythroid cells (due to EPO use), a relative immunosuppressed state and some particular patient populations (e.g. sickle cell disease) lead to an increased theoretical risk of this complication. 

PVB19 may be diagnosed using serology although immunosuppressed patients may not mount an adequate antibody response so PCR viral load is commonly employed. Treatment is non-specific and is generally supportive. IVIg is often used as these pooled preparations have anti-PVB19 antibodies, although randomized controlled data supporting its use is not available. Cutting back immunosuppression in transplant patients may also be of benefit, initially by reducing/stopping the anti-metabolite (logical as mycophenolate frequently induces bone marrow suppression independently). Also, tacrolimus is considered by some to be particularly conducive to PVB19 infection and a switch to cyclosporine may be a next step. 
To summarize, PVB19 is important to the nephrologist from a clinical perspective particularly for its tendency to cause isolated severe anemia (or pancytopenia) in our immunosuppressed patients. It may also cause glomerular disease in immunocompetent patients and should be a differential diagnosis for otherwise unexplained glomerulopathy, especially collapsing FSGS. It also provides clues from a research perspective to mechanism of glomerular disease/sclerosis.

Tuesday, July 8, 2014

Lessons from the Akita mouse

Diabetic nephropathy (DN) is a major cause of ESRD worldwide. While many attempts have been made to develop reliable animal models that mimic human disease—ob/ob, db/db obese diabetes type 2 diabetes models, NOD1 mice, streptozotocin (STZ)-induced diabetes model etc., current mouse models still do not display full spectrum of functional and pathological process of human DN (JASN 2009). In addition, it was revealed recently that genetic background has an important effect on the development and severity of diabetic nephropathy. Indeed, C57BL/6 strain, most commonly used for many experimental studies, is highly resistant to diabetic injury. Recently, Akita mouse gained great interest in research of DN. Let’s start with some history of Akita mouse.

Akita mouse was initially reported as a mouse model mimicking MODY (maturity onset diabetes of the young) in late 1990s (Diabetes 1997). Later, they were found to exhibit type 1 diabetes mellitus via a spontaneous point mutation (C96Y) in the Ins2 gene (Wang JCI 1999), which disrupts a disulphide bond between the insulin A and B chains in a dominant negative way, resulting in misfolding and accumulation of insulin molecules (a.k.a ER stress). Akita C57BL/6 develops spontaneous hyperglycemia at 400-500 mg/dl range, mild hypertension, and albuminuria approximately 50-100 microgram/ day, at around 3-4 weeks of age. Difference in disease susceptibility, especially in severity of albuminuria, depending on genetic backgrounds was reported. (Gurley, Am J physiol Renal Physiol 2010, Methods Mol Biol 2012)

Here are recent research updates on Akita mice:

 KKS (Kallikrein-kinin system)—bradikinin 1/2 receptor deficiency in Akita mouse
ACE inhibitors exhibit renoprotective effects via decrease in intra-glomerular pressure by dilating efferent arterioles. Another explanation of renoprotective effects of ACE inhibitors is via kallikrein-kinin system (KKS). B1R and B2R are the two bradikinin receptors; B2R expresses constitutively and, on the other hand, B1R is inducible by inflammatory stress and DN. Studies by Kakoki et al. (Kakoki PNAS 2004 and PNAS 2010) showed B1R and B2R KO mice developed more severe kidney albuminuria (1.7 x (B2R KO-Akita) and 3.0 x (BR double KO-Akita) compared to Akita WT) and glomerular pathology at 6 or 12 months of age, suggesting potential renoprotective effects of KKS. Hypothesis is that the lack of B2R/B1R enhances oxidative stress via reduction of eNOS and prostaglandins as well as mitochondorial dysfunctions. More details to follow.
 eNOS (endothelial Nitric Oxide) deficiency in Akita mice 
In humans, three variants in the endothelial NOS (eNOS) gene NOS3—G894T in exon 7, tandem repeats in intron 4, and C786T in the promoter—are associated with DN. Actually, the frequency of G894T is relatively common and 5-9% individuals are homozygous for TT and thus with less activity of eNOS. Wang et al. (Wang et al. PNAS 2011) developed eNOS-/-Akita mouse in B6-129 hybrid background (eNOS-/-Akita in C57BL/6 is lethal around 5 months before developing DN), and showed eNOS KO resulted in increase in glomerular filtration (increase at 3 mo, then decrease at 7 mo), basement membrane thickening, glomerulosclerosis and albuminuria, independent of blood glucose and blood pressure. 
 Other models in Akita mouse
ACE2 (anigiotensin-conversion enzyme 2) deficiency (Wong et al. Am J physiol 2007) and ketogenic diet (Poplawski et al. PlosONE 2011) in Akita mice have been explored and details to follow.

In summary, Akita mouse is an excellent model of human DN, mimicking both pathophysiology (hyperfiltration and albuminuria) and pathology (GBM thickening and glomerular sclerosis) of kidneys exposed to high serum glucose.

Naoka Murakami

Monday, June 30, 2014

“Liquid Biopsy” – measuring cell-free circulating donor DNA to predict allograft rejection

Transplant physicians face the challenge to detect allograft rejection using non-sensitive biomarkers and clinical signs/symptoms. 
For kidney transplant, serum creatinine, urinalysis for proteinuria or hematuria, are so far used for screening for rejection and/or assessment of graft function. In a recent article in Sci Transl Med, De Vlaminck et al. investigated the use of cell-free circulating DNA (cfdDNA) as a marker of allograft rejection in adult heart transplants. 
Authors used shotgun sequence approach to detect donor-specific DNA fragments (SNP genotyping), and correlated the level of cfdDNA with cardiac biopsy results, the diagnostic “gold standard” for rejection. 
The idea of a “liquid biopsy” —using plasma circulating DNA fragments—as a diagnostic tool has been explored in the Oncology field, to detect early and late stage cancers. In transplant field, the presence of donor-derived DNA fragments in recipient’s plasma (or urine), called microchimerism, has been known since the end of 1990s  and using them as a measure of transplant injury has been tried. For example, Sidgel et al. used Y-specific DNA fragments as a marker in male-to-female transplants. 
 In this current article, De Vlaminck et al. further improved this approach to overcome dependency on donor-recipient pair. Authors concluded that using the cutoff value of 0.25%, cfdDNA method is as sensitive (0.58) and specific (0.87) as biopsy (0.58 and 0.96, respectively). Furthermore, cfdDNA approach was able to detect rejection as early as 5 months prior to tissue diagnosis
There are, of course, some limitations to overcome: 1) cfdDNA cannot differentiate ACR vs AMR, 2) cannot rule out other source of DNA fragments, such as endothelial damage without rejection 3) exhibit still substantial discordance with biopsy results. Although currently using the shotgun approach, the technique can be refined by using targeted-sequencing approach, to be less laborious process. 
Based on the same underlying principles, it is very likely that similar test would have application in kidney transplantation and potentially help to detect earlier ongoing graft injury. We will still need the biopsy though to clarify the type of injury...

Naoka Murakami

Monday, June 23, 2014

IgM Nephropathy: What You Need to Know

Similar to my recent post on C1q Nephropathy, IgM Nephropathy (IgMN) is a podocytopathy that is often considered a variant of Minimal Change disease (MCD) or FSGS. Like C1QN, it is also a controversial entity with a lack of uniformity from a diagnostic point of view. This undoubtedly leads to under-reporting with some pathologists calling a case IgMN what others would call MCD/FSGS/Mesangial Proflierative GN with low level IgM staining.
First descriptions of IgMN reported mesangial hypercellularity on light microscopy, granular IgM and C3 mesangial deposits on immunofluorescence with electron dense deposits in about half of cases. Podocyte foot process effacement was also evident consistent with the presentation of nephrotic syndrome. The incidence has been reported to be 2-18% of renal biopsy series and age at presentation appears to be bimodal with a peak in childhood and again later in life, 6-7th decades. The incidence of IgMN may be higher in the developing world and most recent reports come from developing nations (ref, ref).
The accurate diagnosis of IgMN is important from a prognostic and therapeutic point of view. Overall, IgMN does not respond to steroids as well as MCD with a higher proportion of patient with steroid-dependent and resistant disease. Furthermore, assessment of children with steroid-dependent and resistant MCD revealed a high proportion of IgMN, which appeared to respond better to cyclosporin than to cyclophosphamide. Rituximab (as ever!) may be another alternative therapeutic choice, although evidence remains at case report level, including a case of recurrent IgMN post-transplant. Similar to C1QN, the severity is likely dependent on the light microscopic changes with MCD patterns reporting an excellent remission rate and an FSGS pattern of injury having a worse prognosis (ref). Most cases however may have mesangial proliferative changes evident. There is even a report of IgMN presenting with crescentic lesions.

IgM deposition may be seen in many other glomerular disorders and its role is unclear. Is it an innocent bystander, reflecting natural IgM binding to exposed epitopes on injured glomerular cells? There is some evidence that IgM deposition may activate complement and that efforts to prevent IgM antibody deposition may prevent complement activation and slow the progression of glomerular injury. IgMN is another glomerular disease that we have an unclear understanding of pathogenesis and etiology where we rely on crude pathological descriptions. Hopefully the future will bring clarity to these diseases and facilitate precise molecular diagnosis of entities such as IgMN.

Sunday, June 22, 2014

POSEIDON Trial for Contrast Induced Nephropathy: The Next #NephJC

The next Nephrology Twitter Journal Club (@NephJC) will discuss the POSEIDON Trial which has been published in The Lancet. Interest in the prevention of Contrast Induced Nephropathy (CIN) is alive and well with several novel approaches/therapies being recently reported (see my recent post), mostly in non-Nephrology journals. Despite these interventions, volume expansion remains the cornerstone of preventative management for CIN (contrast-induced acute kidney injury is just too much of a mouthful for me).

This current trial randomized patients into 2 protocols of 0.9% NaCl-based volume expansion peri-cardiac catheterization. The study group had their fluid rate governed by left ventricular end diastolic volume, measured easily during the catheterization procedure and used as a surrogate for volume status. This allowed the intervention group patients to safely receive higher volumes of saline during their procedures which translated into significantly lower rates of CIN. Obviously this method can only be conveniently employed during catheterization procedures and would need the involvement of interested cardiologists for it to be used in real-life clinical practice. However, the results appear impressive.
You can read a concise summary of the trial and results over at NephJC. Be sure to tune into Twitter on Wednesday June 25th at 9pm Eastern and feel free to get involved using #NephJC.

Monday, June 16, 2014

Exams Are Coming

This season's Game of Thrones finished last night and as a nephrology-themed homage, a trainee from Malaysia, Lee Jun, created this poster during downtime while studying for his exams. Enjoy.

Saturday, June 14, 2014

C1q Nephropathy: What You Need to Know

I saw a 20 year old African American male in clinic with nephrotic range proteinuria, reduced GFR and no signs or symptoms of a systemic disease. His mother developed ESRD at age 36 and his brother, aged 26, has CKD Stage 4. A full laboratory workup including complement proteins and ANA was unremarkable. Renal biopsy demonstrated an FSGS pattern of injury with a full house picture on immunofluorescence but with dominant C1q staining. A diagnosis of C1q Nephropathy (C1QN) was made. 

Jennette et al described C1QN in 1985 in 15 patients without evidence of lupus who had dominant mesangial C1q deposition, often with C3 and Ig’s also. The mean age at presentation was almost 18 years, with a similar male: female ratio and average proteinuria of 7.5g/day. Another series from Columbia University included 19 patients who were predominantly African American (73%), young and female (73%) with nephrotic range proteinuria. Renal outcomes across the series’ (including this European series) are mixed but would suggest immunosuppressive treatment directed against the primary pathology should be considered.
C1QN may be considered an independent pathological entity, although this has been controversial, with some classing it as a subtype of FSGS or minimal change disease (MCD). The prevalence varies (0.2-16%) but is more common in children. The predominant light microscopic appearance may be MCD, FSGS or a proliferative glomerulonephritis. While C1q deposition (predominantly in the mesangium; see image) is dominant or co-dominant by definition, a full house pattern of IgM, IgG, IgA, C1q and C3 may be observed, more commonly with a proliferative glomerulonephritis. As for treatment, the case series are small and largely uninformative. Some patients appear to remit with or without immunosuppressive treatment whereas others have a rapid course towards ESRD. Patients with an FSGS pattern may do worse than other types. For what it’s worth, my patient had no response to 6 months of steroids and mycophenolate and a plan for cyclosporine was made. The fact he likely has a genetic element to his disease adds complexity. Some would argue that immunosuppressive therapy in these cases is not likely to be of benefit but we see from our hereditary FSGS cohort that this is not necessarily the case. Cyclosporine and Rituximab in particular appear to have a directly beneficial effect on podocyte function (also see case report of Rituximab treatment in C1QN).
As for the pathogenesis and significance of the C1q deposits, we have much to learn. Are the C1q deposits pathogenic or merely markers of injury? We do know that C1q binds to the basement membrane via laminin and C1q receptors enhance binding of immune complexes to mesangial cells where it combines with other complement proteins to form C1 protease. This in theory allows activation of the classical complement cascade. Despite this, serum C4 levels are generally normal and I can find no reports of complement directed therapy (Eculizumab or C1-Inhibitors) in C1QN.
Bottom line: C1QN is likely an under recognized entity which can have varying clinical and histological presentations. Renal outcomes are also variable and it may have a different prognosis compared to traditional MCD (& FSGS?). One to keep in mind for the boards.

Friday, June 13, 2014


A few months ago, a paper was published in the International Journal of Epidemiology that caused a sensation in it's home country - Denmark. Using registry data, and the diagnosis of non-melanomatous skin cancer as a proxy for sun exposure, the authors found that the OR for mortality in individuals who developed skin cancer compared to those who did not was 0.53. The p-value was an unfeasibly low 2x10E-308. However, when the results were stratified by age, the OR was a much more reasonable 0.97 in the sun-exposed group. This fact was conveniently forgotten when the study was reported in the media which uncritically stated that people who get more sun will live longer - something which upset the Danish cancer societies immensely.

So why was there such a disparity between the two results and how could it be possible that individuals with cancer could live so much longer (8.5 years on average) than those who did not get cancer. This month, a mea culpa editorial was published in IJE explaining their mistake along with an article discussing the entire issue. What the authors did in this case was fail to account for the immortal time bias. In this study, individuals entered the cohort when they were 40 years old. However, most people did not develop skin cancer until they were in their 60s or 70s. As a result, for the skin cancer cohort, there were approximately 20 years where they could not have died (they had to survive until they developed cancer and were "immortal" until that time). Someone who did not develop skin cancer could have died at any time during that 20 year period. To demonstrate how this works, the authors of the follow-up study, using the same dataset, randomly allocated a "lottery prize" to a proportion of individuals with a mean age of 68 who lived in Denmark over the last 20 years. Again, in this case, a person who received the prize would have had to have lived until the time that it was awarded while those who did not get the prize could die at any time. The results of the simulation study were very similar to the skin cancer study with an OR for all-cause mortality of 0.5 for the prizewinners and a similarly outrageous p-value.

This issue was first described in the 1800s when it was noticed that generals and bishops live longer than lieutenants and curates. Again, this is because one has to survive to an older age to become a bishop or a general and not because there is something inherent in these ranks that lead to an improvement in mortality.

This example is particularly egregious and the editors of IJE have to be congratulated for the way that they dealt with it. However, there are more subtle examples, one of which, highlighted in the follow-up article, appeared in JASN in 2010. This paper found that survival after transplant failure was improved by nephrectomy. However, the mean follow-up in this paper was only 2.93 years and the mean time to nephrectomy was 1.66 years. Thus, about half of the follow-up in individuals who had a nephrectomy was immortal time - they could not die during that follow-up period because they had to survive to the time of surgery. Most of the difference could be accounted for by this bias.

Similarly, a study in JASN in 2007, found that individuals enrolled in a multidisciplinary care clinic were more likely to survive than those who were not. Again, the time of the first MDC clinic was about 1 year after enrollment in the study. Individuals who entered the MDC program had a year of immortal time compared to individuals who did not enter MDC. This was pointed out in a follow-up article in KI. The authors of the original paper reanalyzed their data to account for this bias and found that MDC was still associated with improved survival although the magnitude of the effect was substantially less.

This is a fascinating issue and probably affects more cohort studies than we think. As a reviewer, I'll certainly try to look out for it in the future.

Wednesday, June 11, 2014

Unusual transplant immunology case

File this under something that we are never likely to see in clinical practice. In 2002 a fascinating case report was published in the New England Journal of Medicine concerning unusual findings during a work-up for a living related kidney transplant. A 52-year old woman had FSGS and was approaching ESRD. Her three sons were willing donors but when they went for HLA typing, there was a problem. Although all of her sons had a HLA haplotype from their father, two of the three had a HLA haplotype which, while identical to each other, did not match either of their mother's HLA haplotypes suggesting that she could not be their mother! Further investigation revealed that her sons' non-paternal HLA haplotype was identical to that of the patient's brother and had ultimately been derived from her deceased father.

Further analysis of the patient's blood found that there was no evidence of two distinct cell populations. However, hair follicles, buccal mucosa, bladder samples from a prior cystoscopy and thyroid cells from a prior resection found 4 haplotypes in these tissues. Only two were present in her peripheral blood. There was a normal number of chromosomes in all cells. This was a case of tetragametic chimerism. It is likely that there were two fertilized embryos in utero and that they fused at some point during development to form a single individual. It turns out that under experimental conditions inducing chimerism in mice, it is not unusual for such individuals to express a single haplotype pair in the peripheral blood while continuing to have all 4 haplotypes in other tissues. No-one really knows how common this condition is although the authors suggest that it may be more common than we appreciate. It has important implications in particular for forensic science.
One of the more interesting aspects of this case was that the patient had a HLA identical brother and a HLA haploidentical brother. Although she appropriately lysed cells from control individuals, she did not react to cells from her brother despite the fact that her lymphocytes carried a different HLA haplotype. She was fully tolerant to her brother.

A couple of years later, a woman in Washington State was in the middle of a paternity suit after separating from the father of her two children. She was pregnant with the third child at the time. Testing revealed that he was indeed the father of the children but that she was not. She was charged with fraud and accused of taking part in a surrogacy scam. Her children were taken off her and when her third child was born, this child was tested too and also found not to be her child. Her lawyer found this case report and asked for further testing - results of a smear test revealed that the DNA in that sample matched her children - she was a chimera also. Her children were promptly returned.

Monday, June 9, 2014

Hyponatremia Guidelines - NephJC

Tomorrow night will be the 4th edition of the Nephrology Journal Club on twitter.The article this week is unusual - it is a clinical practice guideline for the management of hyponatremia that was published in NDT, the European Journal of Endocrinology and Intensive Care Medicine. It is an interesting piece of work that has been expertly summarized by Joel Topf over at PBFluids. Even if you do not feel up to reading the entire paper, consider having a look at his executive summary as it raises a number of important (and eminently debateable) points.

See also the summaries at and Nephron Power. Join everyone for what is likely to be a lively discussion tomorrow at 9pm ET on twitter using the hashtag #NephJC.