Diabetes and CKD - Pitfalls: Cystatin C

Cystatin C has been proposed as an alternative marker of kidney function and studies have shown that CyC is a better predictor of mortality that serum creatinine. Although, when first introduced, it was thought that CyC was not influenced by factors apart from renal function, this assumption has been questioned in the recent past.

CyC is a 13 kDa cysteine protease inhibitor that is produced by all nucleated cells. It is freely filtered at the glomerulus and then catabolized in the proximal tubule such that very little appears in the urine. CyC levels are affected by renal function but also independently influenced by age, gender, BMI, fat mass, triglycerides and the presence of diabetes. Interestingly, these are all components of the metabolic syndrome.

In 2011, a paper was published in Diabetologia that found that elevated levels of CyC were associated with an increased incidence of type II diabetes. The thought was that CyC was potentially involved in the pathogenesis of diabetes. In July, a paper was published in NDT that shed a bit more light on this issue. The authors reported the results of a 3-year study of French adults in whom the incidence of diabetes was low. In this study, in common with previous research, CyC predicted incident diabetes. However, when stratified by BMI, CyC predicted incident diabetes only in participants with a BMI >25 at baseline.

So what is the explanation for this? CyC secretion has been shown to be 2-3 times higher in obese patients than in non-obese patients. CyC is also highly expressed in subcutaneous adipose tissue. Data from the Framingham Heart Study has shown that adipose tissue was not associated with CKD using creatinine-based estimating equations while it was associated with CKD using a CyC-based equation. CyC may play a role in preventing inflammation associated with increased adiposity explaining the increased secretion in obese patients.

The implications of this are that, although CyC may predict diabetes, it is unlikely that it adds any more to prediction algorithms considering that it is not independent of BMI and the metabolic syndrome - both of which are well known to be associated with diabetes. The second implication is that the fact that CyC is better at predicting mortality than creatinine (at the same level of eGFR) is related to non-renal factors - again, adiposity and the metabolic syndrome. It similarly suggests that in obese patients, estimating equations that utilize CyC may not be as accurate as previously suggested. The search for a better biomarker of GFR continues...

Diabetes and CKD - Pitfalls: Estimating GFR

The routine use of estimating equations for GFR has revolutionized the way that we view renal disease over the last 15 years and although some argue that this has lead to overdiagnosis of CKD, I believe that this has been an extremely positive development both in clinical and research terms. One criticism of the MDRD equation in particular was that it did not perform well in patients with near normal GFR and the CKD-Epi equation was introduced, at least in part, because of this limitation. However, there remain concerns that in patients with diabetes, particularly in those with hyperfiltration, this formula still does not perform sufficiently well.

To answer this question researchers in Italy took patients from two clinical trials who had serial measured GFR for up to 8 years and compared the results with simultaneous estimates of GFR using the 14 different equations. Of the 600 patients included, 15% were hyperfiltering and 13% had a reduced GFR. Overall, all but one of the equations underestimated GFR in the group as a whole. The single equation that overestimated GFR (Ibrahim) tended to overestimate at all levels. The range of differences between the mGFR and eGFR was -40 to +20 ml/min/1.73m2 and the mean percent error (MPE) ranged from -28.14 to 0.98%. Not unexpectedly, the majority of the error was related to underestimation of GFR in patients with hyperfilatration (MPE -12.8 to -36.7%). It is notable that the MPE was lowest in participants with hyperfiltration using the CKD-Epi equation. In this group, the mean mGFR was 132 ml/min/1.73m2 while the mean eGFR ranged from 83-114 ml/min/1.73m2.

The bias was far lower for the normofiltration and low GFR groups. Because the authors had longitudinal data also, they were able to look at the ability of the formulas to measure GFR decline over time. Given that all of the equations underestimated GFR at baseline, it is unsurprising that there was systematic underestimation of GFR decline over time, particularly in the patients with hyperfiltration. This was less marked in the patients with CKD at baseline. Five of the equations actually estimated that GFR was increasing in the patients despite a consistent decline in mGFR.

This is all not to say that these formulas are not useful. It is always important to recognize the limitations of your tools and one of the major issues here is that creatinine is used as the marker of kidney function with all of the limitations that this introduces. It should also be said that although the agreement with mGFR might not be great, we know from large EPI studies that an eGFR of less than 60 ml/min/1.73m2 is associated with poorer outcomes and this is true no matter what the cause of the disease. The take home from this is that it is not possible to accurately diagnose hyperfiltration in diabetic patients without over nephropathy using current creatinine-based estimating equations and that other signs should be taken into account when assessing these patients.

(Click on images to enlarge)

Don't Eat the Leaves

Hyperoxaluria is an important risk factor for kidney stones, approximately 80% of which are primarily composed of calcium oxalate. Hyperoxaluria is typically diagnosed by performing a 24 hour urine collection and levels above 45 mg/day are considered abnormal although, depending on the other urine constituents, the risk of CaOx stones increases when the urinary oxalate level is above 20 mg/dl. It is important to distinguish between hyperoxaluria that results from increased oxalate production (endogenous) and increased oxalate ingestion (enteric).

The classic disease associated with increased oxalate production is primary hyperoxaluria. There are 3 identified types although all result from defects in glycoxylate metabolism leading to oxalate accumulation. At first, the manifestations are primarily renal leading to nephrolithiasis and nephrocalcinosis. However, as the disease progresses,  the serum oxalate concentration increases eventually resulting in extra-renal oxalate deposition. Vitamin C is metabolized to oxalate also so that patients with oxalate-containing kidney stones should probably avoid excess vitamin C supplementation as this could increase the risk of stones.

Enteric hyperoxaluria results from increased absorption of oxalate in the large bowel. In general, there are 3 ways in which this might occur:

• Increased dietary oxalate ingestion
• Decreased dietary calcium intake - calcium binds oxalate in the gut and reduces absorption. This is why low calcium diets are not recommended in patients with idiopathic kidney stones. Calcium supplements are a different issue as they may contribute to hypercalciuria and not decrease oxalate ingestion, particularly if they are not taken at mealtimes
• In the setting of malabsorption syndromes and GI disease. This occurs in patients following bariatric surgery, fat malabsorption and inflammatory bowel disease. The mechanism is thought to be related to binding of calcium to fatty acids thus reducing the availability of calcium for oxalate-binding, along with increased large bowel permeability. There have been multiple cases of patients developing severe oxalosis following jejuno-ileal bypass surgery.

The treatment of hyperoxaluria depends on the cause. For all patients, increasing fluid intake is good advice. Some patients with primary hyperoxaluria respond to treatment with pyridoxine which promotes conversion of glycoxylate to glycine instead of oxalate. Recently, a bacterium has been identified that metabolizes oxalate in the gut and this has been proposed as a potential treatment for hyperoxaluria. Interestingly, antibiotic treatment has been shown to decrease oxalobacter colonization in individuals with peptic ulcer disease.

Of course, all patients with hyperoxaluria should be advised to reduce oxalate consumption in the diet. Foods high in oxalate include spinach, rhubarb, tea, chocolate, star fruit and soy products. A full list can be found here.

Rhubarb is an interesting case. In the First World War because of the lack of access to fresh vegetables, the British government recommended that families supplement their diets with rhubarb leaves which were not traditionally eaten. It turns out that this was very bad advice. Rhubarb leaves contain considerably more oxalate than the stalks and there was a flurry of case reports towards the end of the war detailing cases of oxalate poisoning from rhubarb leaf consumption (see also and this). The toxicity of the leaves was probably increased by advice to cool the leaves with soda which increases the solubility of oxalate. Although the MD50 of oxalate would require the ingestion of about 5kg of rhubarb leaves, one could imagine that much lower doses would be toxic in patients with chronic kidney disease.

One last point about oxalate. It is a terminal metabolite and was thought to not have any positive role. However, recent data have suggested that oxalate is important for chloride transport in the proximal tubule where it acts similarly to formate..

Ten Things Every Primary Care Physician Should Know

An article from a few years ago that might provoke some discussion. Some of the recommendations are a little out of date (I don't think I would target a Hb of 12 anymore in patients with CKD) but overall it seems like good advice.

The Top 10 Things Nephrologists Wish Every Primary Physician Knew

1. A "normal" serum creatinine level may not be normal.
2. Know the medications that spuriously raise the serum creatinine.
3. Patients with decreased GFR or proteinuria should be evaluated to determine the cause; positive urine dipstick results for protein should be followed up with a spot urine protein/creatinine ratio.
4.  In patients with early-stage CKD, periodic evaluation and intervention are appropriate to slow the progression of renal disease and avoid complications.
5. Do not automatically discontinue an ACEi or ARB solely because of a small increase in serum creatinine or potassium.
6. Anemia in patients with CKD should be treated with ESAs but should not be over-treated.
7. Phosphate-containing bowel preparation should be used with caution
8. Patients with CKD should avoid magnesium or aluminum-containing oral preparation. Concomitant use of citrate-containing preparations and aluminum-containing oral preparation is hazardous because it can lead to acute aluminum toxicity
9. Although most patients with hypertension should not be screened for secondary hypertension, certain clinical clues may suggest the presence of an underlying cause that, when addressed, may resolve or improve the patient's hypertension
10. In patients with recurrent stone disease, an in depth metabolic evaluation is needed to identify and treat modifiable risk factors, thereby preventing further episodes and/or promoting stone dissolution

I don't see much to disagree with on this list - I might not put the knowledge that bactrim and cimetidine increase creatinine so high on the list. I particularly like number 10 - most patients with recurrent stone disease are unaware that more than 95% of stone recurrences can be prevented with proper medical therapy.

With regard to number 8, although we don't really use aluminum-containing phosphate binders any more, it is still contained in some over-the-counter antacids. Citrate markedly increases the absorption of aluminum from the bowel and in patients with CKD, this has been associated with severe toxicity and death in one case series.

Are there any other things that we should add to this list?

Hypocalcemia - What's the diagnosis - Answer

This was an interesting case. A woman with a previous history of hyperparathyroidism and multiple neck surgeries who developed severe, symptomatic hypocalcemia 3 hours following a parathyroidectomy was thought to have failed because of a persistently elevated PTH level. This suggests that did in fact successfully remove her parathyroid gland and that there was an issue with the assay.

In the hospital referred to in this case, the assay used was a Roche Elecsys PTH STAT assay. This is an antibody-based assay that uses two murine antibodies - a "capture" antibody and a "signal" antibody. In patients who have been previously exposed to murine tissue, anti-mouse antibodies can be present in the serum that bind to both of these antibodies giving a false positive result on the assay. In this case, the patient had received OKT3 in the past which has been associated with a prevalence of anti-mouse antibodies of up to 26%. Her PTH level was repeated using an alternative assay - her pre-op PTH was 18 pg/ml and her post-op PTH was 5 pg/ml. Unfortunately, she required long-term treatment with vitamin D and calcium supplementation. In retrospect, the normal calcium and phosphate in a patient with this degree of apparent hyperparathyroidism and normal kidney function was a clue that there may have been an issue with the test although, of course, it is easy to say that knowing the answer.

Congratulations to "anonymous" for getting the correct answer. The complete case report can be found here.

Ultrasound Course for Nephrologists

Continuing our practice of highlighting excellent courses for Nephrologists and Fellows, here is a message from Professor W. Charles O'Neill regarding the annual Ultrasound for Nephrologists Course at Emory University this October. The course brochure and application form are here. 

"Ultrasonography is an invaluable tool in nephrology and, following the lead of other specialists, nephrologists are beginning to incorporate ultrasound into their practice. It is convenient, economically feasible and improves patient care. Since this skill is not generally taught in training programs, we have been offering the Ultrasonography for Nephrologists course. The course has been very well received and has been given continuously since 1997. It is a comprehensive, weekend program that provides both didactic and hands-on training in ultrasonography of native and transplanted kidneys, end-stage kidneys and allografts, and guidance for biopsy and central venous catheterization. There are 6 hours of hands-on training that include patients with a variety of abnormalities. Also covered are financial aspects including equipment costs, billing, reimbursement, and other aspects such as reports, record-keeping, databases, quality assurance, and accreditation. There will also be an opportunity to use equipment from a variety of manufacturers. Pretty much everything a nephrologist needs to get started."

Hypocalcemia - What's the Diagnosis?

A 36yr old woman with a history of PCKD was referred for surgical treatment of hyperparathyroidism. She had a history of two prior failed kidney transplants following which she had been on dialysis for more than 10 years. She had a third renal transplant 10 years prior to this presentation which was still functional. At the time of her transplant, she had received OKT3 and was currently maintained on tacrolimus and prednisone. She had a long history of hyperparathyroidism. Six years prior to her transplant, she had a subtotal (3.5 glands) parathyroidectomy. The year of her transplant, she had another neck exploration with the removal of 8 nests of hypercellular parathyroid tissue.

At the time of this presentation, her PTH was 3,300 pg/ml despite treatment with cinacalcet. Her ionized calcium was normal (1.2 mmol/L) but she complained of poor concentration, fatigue and depression. Serum phosphorous and creatinine were normal. She had a normal sestamibi scan but an US revealed hyperplastic parathyroid tissue in her neck.

During the parathyroidectomy, frozen section revealed hypercellular parathyroid tissue that was removed. However, pre-op, intra-op and post-op PTH levels were elevated at >1700 pg/ml. Because of the persistently elevated PTH, it was considered a failed operation and calcium supplementation was not started post-op. However, within 3 hours of the operation, she developed perioral numbness, tetany and muscle spasms in her legs. Her ionized calcium was low (0.9 mmol/L) and her symptoms resolved after treatment with iv calcium. Her PTH remained elevated at 3000 pg/ml.

What is the reason for her acute hypocalcemia in this setting? Answer in the comments please.

eJournal Club - Session length and weight gain

This month's eJournal club concerns a paper published by a fellow from our institution that attempts to get to the bottom of an interesting question: in patients with a high interdialytic weight gain (IDWG), is the dialysis session length (DSL) or the total volume gained more important? Both of these have been associated with increased mortality and both lead to an increased ultrafiltration rate (UFR). However, because they are interrelated, it is difficult to say which is more relevant.

For this study, the authors looked at more than 14,000 patients attending dialysis in the US. They excluded patients at extremes of session lengths and those who did not gain any weight between sessions. Patients with a URR less than 65% were also excluded to rule out any effects of underdialysis. The mean IDWG and DSL over 30 days were chosen as the exposures of interest. Interestingly there was high correlation between the 30-day IDWG and DSL and the 60 and 90-day means. The outcome was death from any cause. For the purposes of the analysis, the participants were divided into 2 groups for each exposure - less than or greater than 3kg IDWG and less than or greater than 240 minutes for DSL. A matched case-control study design was used.

Not surprisingly, the patients with higher IDWG tended to be younger, male, AA, had higher blood pressure and a higher prevalence of diabetes and CHF. Patients with lower DSL were more likely to be female, older and were less likely to have diabetes, CHF and CAD.

For the DSL analysis, lower DSL was associated with a HR of 1.32 (1.03-1.69) for mortality after full adjustment. For the IDWG analysis, increased IDWG was associated with a HR of 1.29 (1.01-1.69) for mortality. Thus both DSL and IDWG were independently associated with mortality.

What does this study mean for clinicians. It suggests that targeting both of these interventions could be useful. However it should be pointed out that this is an observational study and that they could not show that changing any of these exposures changed risk. Also, the because of the study design, the authors can only state conclusively that both are associated with mortality and not which one is more important. These conclusions may seem obvious but it is important to have good evidence to present to patients who may be frustrated with our requests to increase times and reduce fluid intake.

Head over the eJournal Club to continue the discussion of this paper.

Renal Jeopardy

Icahn School of Medicine at Mount Sinai are organizing a CME event: "Contoversies in Kidney Disease" aimed at attending nephrologists and fellows on September 13th 2013. This course will cover a broad range of topics of interest to nephrologists including cardio-renal issues, vitamin D and cardiovascular disease, genomics and IgA nephropathy. They have a great group of invited speakers.

In order to sweeten the deal for fellows, the registration fee will be $25 for trainees and there will be a special Renal Fellow Jeopardy Contest during lunchtime.

Kidney Stones - What's the diagnosis? - Answer

This was an interesting case and all those who responded correctly identified that the patient had bowel pathology. However, only one person figured out that the issue was an ileostomy. This patient had a low urine volume and an extremely low urinary citrate and sodium. The low citrate could indicated a renal tubular acidosis except that the urinary ammonium was high and the urine pH was very low indicating preserved ability to acidify the urine. This points to a metabolic acidosis. The urine sodium in an average US resident is between 100-200 mmol/day. Outside of the amazon, it's hard to imagine that anyone could take in this little salt. This points towards loss of sodium bicarbonate and water from the GI tract.

Finally, in the setting of IBD, generally it is accompanied by hyperoxaluria. There are a number of potential mechanisms for this; decreased metabolism of oxalate by oxalobacter formigenes, decreased calcium binding to oxalate because of the relatively increased binding of calcium to malabsorbed fat in the GI tract. In any case, in order to have hyperoxaluria, it is necessary to have a functioning large bowel. In this case, the patient's urinary oxalate was 28 which is in the low normal range and not suggestive of hyperoxaluria. Thus, the diagnosis is high output of alkaline fluid from an ileostomy.

The treatment in this case is to increase fluids and treat with a combination of sodium and potassium citrate. Even a small rise in urine pH would significantly reduce the risk of uric acid stones while the citrate and increased volume should reduce the calcium oxalate stone risk.

Kidney Stones - What's the diagnosis?

A 65yo man was reviewed in the clinic for assessment of kidney stones. He has a history of stones for at least 8 years and has been passing small calculi on a regular basis for the last few months. His 24 hour urine results are shown below (results are 24 hour total values unless otherwise specified):

Volume, Liters	0.71	Sodium, mmol/day	7
Supersaturation Calcium Oxalate	10.11	Potassium, mmol/day	45
Calcium, mg/day	78	Magnesium, mg/day	52
Oxalate, mg/day	28	Phosphate, mg/day	0.76
Citrate, mg/day	11	NH4, mmol/day	68
Supersaturation Calcium Phosphate	0.71	Chloride, mmol/day	48
Urine pH	5.6	Sulphate, mEq/day	39
Supersaturation Uric Acid	2.71	Urea Nitrogen, g/day	11
Uric Acid, mg/day	0.499	Protein Catabolic Rate	1.2
Creatinine	1292

He has a high risk for calcium oxalate and uric acid stones. His urine citrate and sodium are remarkably low and he has a very low urine volume. His BP in the clinic was normal as were his labs apart from a serum creatinine of 1.3mg/dl

What is the underlying diagnosis (an important  piece has been left out of his background history)?
What is the best approach to treating his kidney stones?

Answers in the comments please (if this proves popular we may make it a regular feature).

ASN Annual Meeting Travel Support Opportunities

It's the time of the year again to start thinking about travel to the ASN annual meeting in Atlanta. Thankfully, in this time of limited travel budgets and sequestration, the ASN offers a number of travel awards that will help pay for the cost of attending the conference. For all attendees, if you are participating in a pre-course, there are travel awards available for each of these courses. For fellows in training, there is a specific award that will pay for conference registration and some of the cost of attending.

To encourage medical students and residents to take part, there is a travel award specifically for this category that also offers complimentary ASN membership and $800 towards travel and accommodation costs. Finally, the Mitch Award offers travel support to 3 US fellows who are from underrepresented minorities and 5 fellows from Mexico and Central and South America. Additionally, recipients of this awards will be invited to attend the President's reception.

Needless to say, prior recipients of these awards cannot reapply and for US fellows, at least one year of training must have been completed prior to applying for travel support.

Lonely Glomeruli

One of the difficulties in doing molecular research on the kidney is the diversity highly specialized cells that exist in the glomerulus. As a result, it is important to be able to isolate glomerular tissue from the surrounding kidney. A recent paper in KI detailing a method for isolated podocytes reminded me of a relatively simple technique that I was taught a few years ago for glomerular isolation in mice.

The technique was first described in this paper from 2002 but in brief, it involves injecting the mouse heart with deactivated magnetic beads (after euthanizing them of course). Some of these beads (which are just 5µm in diameter) get trapped in the glomerular capillaries. The kidneys are then removed, minced, digested and passed through a 100µm strainer to remove any larger particulate matter. Finally, the remaining tissue is suspended and exposed to a magnet to pull the glomeruli (with the beads inside) out of the mixture. The glomeruli are then left stuck to the wall of the tube next to the magnet and they can be easily removed.

The picture below is a low-power view of the glomeruli following isolation. You can see that there is very little non-glomerular tissue present, which is remarkable given that the glomeruli make up such a small proportion of renal tissue.

Below is a higher power view of 3 more glomeruli following isolation. You can clearly see the microbeads trapped in the glomerular capillaries. Cool science. (Click on any image to enlarge)

Electrolyte Channels and Aldosteronism

Over the past few years, it has become apparent that hyperaldosteronism is far commoner than was once suspected and screening of unselected patients with hypertension reveals that about 5-10% of patients have primary hyperaldosteronism. In patients with resistant hypertension, that percentage increases to 15-20%. About 30% of hyperaldosteronism is caused by aldosterone producing adrenal adenomas (APA). Most of the rest is related to bilateral adrenal hyperplasia with less than 5% of cases being familial. The secretion of aldosterone in adrenal cells is dependent on the intracellular calcium concentration and increases in response to higher plasma calcium. Entry of calcium into the cells is in turn dependent on voltage-gated membrane calcium channels (which allow calcium influx when the cells are depolarized) and a calcium ATPase which removes calcium from the cells. Under normal circumstances, adrenal cells are hyperpolarized thus keeping these calcium channels closed. Cell polarization is maintained by a combination of the action of the Na-K-ATPase (which exchanges 3 intracellular Na for 2 extracellular K) and membrane K channels lead to K loss from the cells.

Angiotensin II inhibits the Na-K-ATPase leading to cell depolarization, calcium influx into cells and aldosterone secretion. Similar effects are seen when cells are treated with oubain, a specific Na-K-ATPase inhibitor that also leads to hyperaldosteronism.

In 2011 in a seminal paper in Science, Choi et al reported finding somatic mutations in KCNJ5, a membrane potassium channel in patients with APA. These were identified by sequencing tissue from the tumors and comparing with the surrounding tissue. Subsequently, it has been found that about 30-40% of patients with APA have somatic mutations in KCNJ5. These mutations are believed to reduce the ion selectivity of the channels, allowing Na to move into the cell and reduce the resting membrane potential. A number of families have been identified with KCNJ5 mutations resulting in bilateral hyperplasia - now called Familial Hyperaldosteronism type III.

Recently, a paper was published in Nature Genetics which attempted to determine if there were other somatic mutations in patients with APA. In this study, they took KCNJ5-normal patients and sequenced the exons of the tumors and the surrounding tissue. There were very few mutations identified but 5/9 patients had mutations in ATP1A1, a component of the Na-K-ATPase or ATP2B3, a component of the calcium ATPase that removes calcium from adrenal cells. Follow-up targeted sequencing of 300 patients with APA revealed that about 7% had mutations in one of these two genes. Patients with these mutations had higher aldosterone levels, lower minimum potassium levels and higher systolic BP, all indicators of more severe disease. Notably, no families have been identified with these mutations. In vitro studies revealed that cells with these mutations have very low membrane potentials and it is speculated that if this was a germline mutation, it would likely not be compatible with life. This is a fascinating insight into how very small changes in electrolyte channels can have far-reaching consequences and shows a great progression from exome sequencing to the bench and to clinical investigation.

The images in this post are taken from the recent paper in Nature Genetics. One would wonder if somatic mutations explain some of the missing heritability that were are seeing in genetic studies of common diseases. See this previous post by Lisa on the genetic causes of hypertension.